Compositions and methods for characterizing a microbiome

ABSTRACT

A system is provided comprising a plurality of C. elegans cultures, where each culture comprises a transgenic C. elegans strain that models a mammalian disease or condition. Methods of using a system, e.g., for characterizing microbial strains of a mammalian microbiome and determining whether such microbial strains affect a mammalian disease or disorder.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.17/019,183, filed Sep. 11, 2020, which claims priority to U.S.provisional patent application No. 62/988,132, filed Mar. 11, 2020, andU.S. provisional patent application No. 62/899,718, filed Sep. 12, 2019,the entire contents of all of which are hereby incorporated by referencein their entirety.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named “2013404-0024_ST25.txt”on Apr. 29, 2022). The .txt file was generated on Oct. 7, 2020, and is7,072 bytes in size. The entire contents of the Sequence Listing areherein incorporated by reference.

BACKGROUND

Caenorhabditis elegans is a bacteriovorus nematode that is about 1 mm inlength and lives in temperate soil environments.

SUMMARY

The present disclosure provides an insight that C. elegans can provideand/or represent surprisingly useful systems for assessing one or morefeatures of a microbial preparation (e.g., of a microbiome sample).Among other things, the present disclosure describes technologies thatcan be useful for assessing microbiome samples to identify orcharacterize effects and/or modulation of microbial strains of suchmicrobiome samples on certain diseases or conditions. In someembodiments, such technologies can be useful to discern strain-leveldifferences in a particular patient or patient population. Accordingly,the present disclosure also provides technologies that can be useful toassess the nature of the microbial strains in patient-specific samplesand thus provide patient-specific information on how individualpatients' microbiomes differentially affect their health conditions. Forexample, in some embodiments, technologies provided herein can be usefulto identify diseases or conditions to which a patient might besusceptible, based on the nature of the microbial strains inpatient-specific samples. In some embodiments, technologies providedherein can be useful to identify microbial strains in patients that arebeneficial, e.g., to protect patients from or confer resistance tocertain diseases or conditions. Thus, technologies described herein areuseful as diagnostic tools for screening microbiome samples (e.g., humanmicrobiome samples) for disease modifiers (e.g., microbial strains thataffect a disease or condition).

Indeed, in certain embodiments, the use of transgenic C. eleganswhole-animal model systems to identify or screen for microbiome strains(e.g., present in human microbiome) that can modulate or affectpathogenesis and/or development of a neurodegenerative disease orcondition (e.g., Alzheimer's disease). Those skilled in the art readingthe present disclosure will understand that technologies describedherein are applicable for use not only relating to neurodegenerativediseases, such as Alzheimer's disease as exemplified, but also relatingto various other diseases or conditions that may be associated withmicrobiomes, such as, but not limited to Parkinson's disease,Huntington's disease, and amyotrophic lateral sclerosis, type IIdiabetes, obesity, hyperglycemia, glucose intolerance, insulinresistance (i.e., hyperinsulinemia, metabolic syndrome, syndrome X),hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia(e.g., dyslipidemia), hypertriglyceridemia, cardiovascular disease,atherosclerosis, peripheral vascular disease, kidney disease,ketoacidosis, thrombotic disorders, nephropathy, diabetic neuropathy,diabetic retinopathy, retinopathy of prematurity, age-related maculardegeneration, glaucoma, sexual dysfunction, dermatopathy, dyspepsia,hypoglycemia, metabolic syndrome, cancer or edema.

In some aspects, provided herein are systems comprising a plurality ofC. elegans cultures, wherein each culture comprises a transgenic C.elegans strain that models a disease or condition (e.g., a mammaliandisease or condition). In some embodiments, such a plurality of C.elegans cultures may comprise at least 5 or more (including, e.g., atleast 6, at least 7, at least 8, at least 9, at least 10, at least 15,at least 20, at least 25, at least 30, at least 40, at least 50 or more)C. elegans cultures.

In some embodiments, one or more of C. elegans cultures in a providedsystem includes a transgenic C. elegans strain that models a disease orcondition present in a target subject. In some embodiments, one or moreof C. elegans cultures in a provided system may include a transgenic C.elegans strain that models a mammalian disease or condition (e.g., ahuman disease or condition). An exemplary human disease or condition tobe modeled by C. elegans strains may include a neurodegenerative diseaseor disorder (e.g., Alzheimer's disease). In some embodiments, a diseaseor condition (e.g. human disease or condition) to be modeled by C.elegans strains may include a disease or condition associated with analtered or defective HIF-1 pathway (e.g. may comprise a cellular stressresponse). In some embodiments, for example, the disease or condition tobe modeled by C. elegans may be an ocular neovascular disease ordisorder (e.g., diabetic retinopathy, retinopathy of prematurity,age-related macular degeneration, or glaucoma). In some embodiments, oneor more of C. elegans cultures in a provided system may include atransgenic C. elegans that models a non-human mammalian disease orconditions, e.g., a canine, a feline, an equine, a bovine, an ovine, acaprine, or a porcine disease or condition.

In some embodiments, transgenic C. elegans strains provided in a systemdescribed herein may comprise a transgene comprising a characteristicsequence element associated with a target disease or condition (e.g., amammalian disease or condition). Such a characteristic sequence elementthat is associated with a disease or condition (e.g., a mammaliandisease or condition) may be or comprise a foreign gene (e.g., amammalian gene), a DNA regulatory element (e.g., a mammalian DNAregulatory element), and/or a mammalian RNA regulatory element. VariousDNA and RNA regulatory elements are known in the art; one skilled in theart will thus understand that in some embodiments, DNA regulatoryelements that are associated with a disease or condition (e.g., amammalian disease or condition) may be or comprise enhancers, promoters,silencers, insulators, locus control regions, and combination thereof.In some embodiments, RNA regulatory elements such as, e.g., untranslatedregions, introns, splice sites, and combinations thereof, which areassociated with a disease or condition (e.g., a mammalian disease orcondition), may be used in accordance with the present disclosure.

Additionally, or alternatively, transgenic C. elegans strains in aprovided system may comprise a transgene comprising a reporter gene.Non-limiting examples of such a reporter gene may include, but are notlimited to, fluorescent, phosphorescent, and/or bioluminescent proteins.

In some embodiments involving C. elegans cultures, at least two or moreof such cultures may each comprise a transgenic C. elegans strain thatmodels the same disease or condition (e.g., a mammalian disease orcondition). In some such embodiments, at least two or more of suchcultures may each comprise a transgenic C. elegans strain that models adifferent biochemical or molecular pathway associated with the samedisease or condition (e.g., a mammalian disease or condition). In someembodiments involving C. elegans cultures, all of such cultures may eachcomprise the same transgenic C. elegans strains that model the samedisease or condition (e.g., a mammalian disease or condition).

In some embodiments involving C. elegans cultures, at least two or moreof such cultures may each comprise a transgenic C. elegans strain thatmodels a different disease or condition (e.g., a mammalian disease orcondition).

Systems described herein can be used to characterize relationshipsand/or effects of microbial preparations (e.g., of a microbiome sample)with certain diseases or conditions in target subjects. Accordingly, insome embodiments, one or more C. elegans cultures in such a systemcomprise microbes of a mammalian microbiome (e.g., a human microbiome).In some embodiments, each of such C. elegans cultures may includemicrobes of a mammalian microbiome (e.g., a human microbiome). In someembodiments, one or more C. elegans cultures in such a system cancomprise microbes of a canine, a feline, an equine, a bovine, an ovine,a caprine, or a porcine microbiome. Microbiomes used in accordance withthe present disclosure may be obtained or derived from target anatomicallocations of mammalian subjects. Examples of such microbiomes mayinclude, but are not limited to cutaneous microbiomes, oral microbiomes,nasal microbiomes, gastrointestinal microbiomes, brain microbiomes,pulmonary microbiomes, and/or urogenital microbiomes.

In some embodiments, microbes in each C. elegans culture can compriseone or more microbial strains. In some embodiments, microbes in eachculture can comprise a single microbial strain.

In some embodiments, one or more of C. elegans cultures can comprise atherapeutic or nutraceutical agent.

Methods for using a plurality of C. elegans cultures to characterize amicrobiome are also provided herein. In some embodiments, a method isfor screening a microbiome of an individual (e.g., a mammal, e.g., ahuman) to determine if a microbial strain or a combination of microbialstrains affects a mammalian disease or disorder. In some embodiments, amethod is for diagnosing an individual (e.g., a mammal, e.g., a human)based on one or more microbial strains in a microbiome of theindividual. In some embodiments, a method is for monitoring a disease orcondition progression in an individual (e.g., a mammal, e.g., a human)based on one or more microbial strains in a microbiome of theindividual.

In some embodiments described herein, a is provided method comprisingadding microbes obtained from a mammalian microbiome to each of C.elegans cultures of a system described herein. In some embodiments,microbes in each C. elegans culture can comprise one or more microbialstrains. In some embodiments, microbes in each such culture can comprisea single microbial strain. Microbiomes used in methods described hereinmay be obtained or derived from target anatomical locations of mammaliansubjects. Examples of such microbiomes may include, but are not limitedto cutaneous microbiomes, oral microbiomes, nasal microbiomes,gastrointestinal microbiomes, brain microbiomes, pulmonary microbiomes,and/or urogenital microbiomes.

In some embodiments, a method may comprise adding a plurality ofmicrobial strains of a mammalian microbiome to a plurality of C. eleganscultures, wherein a different microbial strain is added to each C.elegans culture, and wherein each culture comprises the same transgenicC. elegans strain, and the transgenic C. elegans strain models amammalian disease or condition.

In some embodiments, one or more of C. elegans cultures can comprise atherapeutic or nutraceutical agent. Thus, in some embodiments, a methoddescribed can further comprise adding a therapeutic or nutraceuticalagent to one or more of C. elegans cultures.

In some embodiments, a method described herein can further comprisedetermining one or more parameter values of a transgenic C. elegansstrain in each of C. elegans cultures. In some embodiments, suchparameter(s) of a transgenic C. elegans is/are associated with amammalian disease or condition that such a transgenic C. elegans strainmodels. Exemplary such parameters of transgenic C. elegans may includebiological functions or phenotypes and/or levels and/or activity ofmolecules (e.g., small molecules, proteins, polypeptide, or transcripts)that are associated with a mammalian disease or condition.

In some embodiments, a method may further comprise: (a) determining,before adding a microbial strain to a C. elegans culture, one or moreparameter values of the transgenic C. elegans strain in such a culture,(b) determining, after adding the microbial strain to the C. elegansculture, the same one or more parameter values of the transgenic C.elegans strain in such a culture, and (c) comparing the one or moreparameter values determined before adding the microbial strain with theone or more parameter values determined after adding the microbialstrain.

Technologies described herein, in some embodiments, can be used tocharacterize microbial strain of a human biome associated with a humandisease or condition. Thus, in some such embodiments, a transgenic C.elegans strain involved in systems and methods described herein models ahuman disease or condition. An exemplary human disease or condition thata transgenic C. elegans strain model is Alzheimer's disease. In someembodiments, a disease or condition (e.g. human disease or condition)that a transgenic C. elegans strain models may include a disease orcondition associated with an altered or defective HIF-1 pathway (e.g.may comprise a cellular stress response). In some embodiments, forexample, a disease or condition that a transgenic C. elegans strainmodels may be an ocular neovascular disease or disorder (e.g., diabeticretinopathy, retinopathy of prematurity, age-related maculardegeneration, or glaucoma). In some embodiments, a disease or conditionthat a transgenic C. elegans strain models is diabetic retinopathy,retinopathy of prematurity, age-related macular degeneration, orglaucoma.

Accordingly, certain aspects described herein relate to technologies forcharacterizing a microbial strain of a human biome. For example, oneaspect provides a method, which comprises (a) adding a microbial strainto a C. elegans culture comprising a transgenic C. elegans strain thatmodels Alzheimer's disease, and (b) determining whether the microbialstrain affects one or more parameters of the transgenic C. elegansstrain, wherein such one or more parameters are associated withAlzheimer's disease. In some embodiments, such a transgenic C. elegansstrain may comprise a transgene encoding a human ssApoE4 protein, ahuman Aβ1-42 polypeptide, or a human pseudophosphorylated tau protein.

In some embodiments, such a method further comprises: (a) determining,before adding a microbial strain to a C. elegans culture, one or moreparameter values of the transgenic C. elegans strain in such a culture,(b) determining, after adding the microbial strain to the C. elegansculture, the same one or more parameter values of the transgenic C.elegans strain in such a culture, and (c) comparing the one or moreparameter values determined before adding the microbial strain with theone or more parameter values determined after adding the microbialstrain. Exemplary such one or more parameters include, but are notlimited to, (i) a level of C. elegans paralysis; (ii) a level of amyloidplaques; (iii) a level of tau filaments; (iv) a level ofneuroinflammation; (v) a level of proteasomal function; and/or (vi) acombination thereof.

In another aspect, the present disclosure provides a method ofcharacterizing a microbial strain of a human biome, comprising: (a)adding the microbial strain to a C. elegans culture comprising atransgenic C. elegans strain that models a disease or conditionassociated with an altered or defective HIF-1 pathway, and (b)determining whether the microbial strain affects one or more parametersof the transgenic C. elegans strain, wherein the one or more parametersare associated with an altered or defective HIF-1 pathway. In someembodiments, such a transgenic C. elegans strain may comprise atransgene encoding a human prolyl hydroxylase EGLN, a human HIFtranscription factor, or a human HIFα protein.

In some embodiments, such a method further comprises: (a) determining,before adding the microbial strain to a C. elegans culture, one or moreparameter values of the transgenic C. elegans strain in the culture, (b)determining, after adding the microbial strain to the C. elegansculture, the same one or more parameter values of the transgenic C.elegans strain in the culture, and (c) comparing the one or moreparameter values determined before adding the microbial strain with theone or more parameter values determined after adding the microbialstrain.

Exemplary such one or more parameters include, but are not limited to,(i) a level of neuroinflammation; (ii) a level of proteasomal function;(iii) a level of C. elegans egg-laying rate; and/or (iv) a combinationthereof.

A transgenic C. elegans strain that expresses two or more of (i) a humanssApoE4, (ii) a human Aβ1-42, (iii) a human pseudophosphorylated tau,and (iv) a UbV-GFP proteasomal marker is also within the scope of thepresent disclosure.

The present disclosure, among other things, also describes uses ofprovided transgenic C. elegans strains, systems, and/or methods toscreen a mammalian microbiome for microbial strains that affect amammalian disease or condition. Also, within the scope of the presentdisclosure includes uses of provided C. elegans, systems, and/or methodsto characterize the effect a microbial strain of a mammalian microbiomehas on a mammalian disease or condition. For example, a human microbiomecan be screened/characterized using technologies provided herein inaccordance with the present disclosure.

The present disclosure describes, among other things, compositionscomprising one or more microbial strains. In some embodiments, acomposition provided herein comprises one or more microbial strains froma mammalian microbiome, extracts thereof, and/or components thereof,which have been assessed, identified, characterized or assayed usingtransgenic C. elegans or methods as described herein. In someembodiments, a composition provided herein comprises two or more, threeor more, four or more, five or more, six or more, seven or more, eightor more, nine or more, or ten or more microbial strains from a mammalianmicrobiome, extracts thereof, and/or components thereof, which have beenassessed, identified, characterized or assayed using transgenic C.elegans or methods as described herein.

In some embodiments, a composition provided herein comprises two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, or ten or more microbial strainslisted in TABLE 8 below.

In some embodiments, a composition provided herein comprisesGluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcussp., L. plantarum, Clostridium butyricum, Paenibacillus sp., Veillonellasp., Bifidobacterium, Bacillus subtilis, Acidaminococcus sp., or acombination thereof. In some embodiments, a combination comprises atleast two of, at least three of, at least four of, at least five of, atleast six of, at least seven of, at least eight of, at least nine of, orall of Gluconacetobacter hansenii, Terrisporobacter glycolicus,Coprococcus sp., L. plantarum, Clostridium butyricum, Paenibacillus sp.,Veillonella sp., Bifidobacterium, Bacillus subtilis, and Acidaminococcussp.

In some embodiments, a composition is a pharmaceutical composition. Insome embodiments, a composition is an ingestible item.

The present disclosure describes, among other things, methods comprisingadministering a composition described herein.

In some embodiments, a method of treating a disease or condition in asubject, comprising administering to a subject in need a composition asdescribed herein. In some embodiments, a disease or condition is aneurodegenerative disease or disorder. In some embodiments, a disease orcondition is Alzheimer's disease. In some embodiments, a disease orcondition may be associated with an altered or defective HIF-1 pathway.In some embodiments, a disease or condition may be an ocular neovasculardisease or disorder. In some embodiments, a disease or condition isdiabetic retinopathy, retinopathy of prematurity, age-related maculardegeneration, or glaucoma. The present disclosure describes, among otherthings, uses of compositions described herein. In some embodiments, ause of a composition as described herein is in the treatment of adisease or condition in a subject. In some embodiments, a disease orcondition is a neurodegenerative disease or disorder. In someembodiments, a disease or condition is Alzheimer's disease. In someembodiments, a disease or condition may be associated with an altered ordefective HIF-1 pathway. In some embodiments, a disease or condition maybe an ocular neovascular disease or disorder. In some embodiments, adisease or condition is diabetic retinopathy, retinopathy ofprematurity, age-related macular degeneration, or glaucoma.

These, and other aspects encompassed by the present disclosure, aredescribed in more detail below and in the claims.

Definitions

The scope of the present invention is defined by the claims appendedhereto and is not limited by certain embodiments described herein. Thoseskilled in the art, reading the present specification, will be aware ofvarious modifications that may be equivalent to such describedembodiments, or otherwise within the scope of the claims. In general,terms used herein are in accordance with their understood meaning in theart, unless clearly indicated otherwise. Explicit definitions of certainterms are provided below; meanings of these and other terms inparticular instances throughout this specification will be clear tothose skilled in the art from context.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

The articles “a” and “an,” as used herein, should be understood toinclude the plural referents unless clearly indicated to the contrary.Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context. In some embodiments, exactly one member of agroup is present in, employed in, or otherwise relevant to a givenproduct or process. In some embodiments, more than one, or all groupmembers are present in, employed in, or otherwise relevant to a givenproduct or process. It is to be understood that the inventionencompasses all variations, combinations, and permutations in which oneor more limitations, elements, clauses, descriptive terms, etc., fromone or more of the listed claims is introduced into another claimdependent on the same base claim (or, as relevant, any other claim)unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists (e.g., in Markush group orsimilar format), it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where embodiments oraspects are referred to as “comprising” particular elements, features,etc., certain embodiments or aspects “consist,” or “consist essentiallyof,” such elements, features, etc. For purposes of simplicity, thoseembodiments have not in every case been specifically set forth in somany words herein. It should also be understood that any embodiment oraspect can be explicitly excluded from the claims, regardless of whetherthe specific exclusion is recited in the specification.

Administration: As used herein, the term “administration” typicallyrefers to the administration of a composition to a subject or system toachieve delivery of an agent to the subject or system. In someembodiments, the agent is, or is included in, the composition; in someembodiments, the agent is generated through metabolism of thecomposition or one or more components thereof. Those of ordinary skillin the art will be aware of a variety of routes that may, in appropriatecircumstances, be utilized for administration to a subject, for examplea human. For example, in some embodiments, administration may be ocular,oral, parenteral, topical, etc. In some particular embodiments,administration may be bronchial (e.g., by bronchial instillation),buccal, dermal (which may be or comprise, for example, one or more oftopical to the dermis, intradermal, interdermal, transdermal, etc.),enteral, intra-arterial, intradermal, intragastric, intramedullary,intramuscular, intranasal, intraperitoneal, intrathecal, intravenous,intraventricular, within a specific organ (e. g. intrahepatic), mucosal,nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g.,by intratracheal instillation), vaginal, vitreal, etc. In manyembodiments provided by the present disclosure, administration is oraladministration. In some embodiments, administration may involve only asingle dose. In some embodiments, administration may involve applicationof a fixed number of doses. In some embodiments, administration mayinvolve dosing that is intermittent (e.g., a plurality of dosesseparated in time) and/or periodic (e.g., individual doses separated bya common period of time) dosing. In some embodiments, administration mayinvolve continuous dosing (e.g., perfusion) for at least a selectedperiod of time. Administration of cells can be by any appropriate routethat results in delivery to a desired location in a subject where atleast a portion of the delivered cells or components of the cells remainviable. A period of viability of cells after administration to a subjectcan be as short as a few hours, e.g., twenty-four hours, to a few days,to as long as several years, i.e., long-term engraftment. In someembodiments, administration comprises delivery of a bacterial extract orpreparation comprising one or more bacterial metabolites and/orbyproducts but lacking fully viable bacterial cells.

Analog: As used herein, the term “analog” refers to a substance thatshares one or more particular structural features, elements, components,or moieties with a reference substance. Typically, an “analog” showssignificant structural similarity with the reference substance, forexample sharing a core or consensus structure, but also differs incertain discrete ways. In some embodiments, an analog is a substancethat can be generated from the reference substance, e.g., by chemicalmanipulation of the reference substance. In some embodiments, an analogis a substance that can be generated through performance of a syntheticprocess substantially similar to (e.g., sharing a plurality of stepswith) one that generates the reference substance. In some embodiments,an analog is or can be generated through performance of a syntheticprocess different from that used to generate the reference substance.

Approximately: As applied to one or more values of interest, includes toa value that is similar to a stated reference value. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within ±10% (greater than or less than) of the statedreference value unless otherwise stated or otherwise evident from thecontext (except where such number would exceed 100% of a possiblevalue).

Comparable: As used herein, the term “comparable” refers to two or moreagents, entities, situations, sets of conditions, subjects, etc., thatmay not be identical to one another but that are sufficiently similar topermit comparison therebetween so that one skilled in the art willappreciate that conclusions may reasonably be drawn based on differencesor similarities observed. In some embodiments, comparable sets ofconditions, circumstances, individuals, or populations are characterizedby a plurality of substantially identical features and one or a smallnumber of varied features. Those of ordinary skill in the art willunderstand, in context, what degree of identity is required in any givencircumstance for two or more such agents, entities, situations, sets ofconditions, etc. to be considered comparable. For example, those ofordinary skill in the art will appreciate that sets of circumstances,individuals, or populations are comparable to one another whencharacterized by a sufficient number and type of substantially identicalfeatures to warrant a reasonable conclusion that differences in resultsobtained or phenomena observed under or with different sets ofcircumstances, individuals, or populations are caused by or indicativeof the variation in those features that are varied.

Conservative: As used herein, refers to instances when describing aconservative amino acid substitution, including a substitution of anamino acid residue by another amino acid residue having a side chain Rgroup with similar chemical properties (e.g., charge or hydrophobicity).In general, a conservative amino acid substitution will notsubstantially change the functional properties of interest of a protein,for example, the ability of a receptor to bind to a ligand. Examples ofgroups of amino acids that have side chains with similar chemicalproperties include: aliphatic side chains such as glycine (Gly, G),alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine(Ile, I); aliphatic-hydroxyl side chains such as serine (Ser, S) andthreonine (Thr, T); amide-containing side chains such as asparagine(Asn, N) and glutamine (Gln, Q); aromatic side chains such asphenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W);basic side chains such as lysine (Lys, K), arginine (Arg, R), andhistidine (His, H); acidic side chains such as aspartic acid (Asp, D)and glutamic acid (Glu, E); and sulfur-containing side chains such ascysteine (Cys, C) and methionine (Met, M). Conservative amino acidssubstitution groups include, for example, valine/leucine/isoleucine(Val/Leu/Ile, V/L/I), phenylalanine/tyrosine (Phe/Tyr, F/Y),lysine/arginine (Lys/Arg, K/R), alanine/valine (Ala/Val, A/V),glutamate/aspartate (Glu/Asp, E/D), and asparagine/glutamine (Asn/Gln,N/Q). In some embodiments, a conservative amino acid substitution can bea substitution of any native residue in a protein with alanine, as usedin, for example, alanine scanning mutagenesis. In some embodiments, aconservative substitution is made that has a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet, G. H. et al., 1992,Science 256:1443-1445, which is incorporated herein by reference in itsentirety. In some embodiments, a substitution is a moderatelyconservative substitution wherein the substitution has a nonnegativevalue in the PAM250 log-likelihood matrix.

CONSERVATIVE AMINO ACID SUBSTITUTIONS For Amino Acid Code Replace WithAlanine A D-ala, Gly, Aib, β-Ala, Acp, L-Cys, D-Cys Arginine R D-Arg,Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-OrnAsparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid DD-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys,Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp,D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln GlycineG Ala, D-Ala, Pro, D-Pro, Aib, β-Ala, Acp Isoleucine I D-Ile, Val,D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val,AdaA, AdaG, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg,D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met,S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr,D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4 or 5-phenylproline,AdaA, AdaG, cis-3,4 or 5-phenylproline, Bpa, D-Bpa Proline P D-Pro,L-I-thioazolidine-4-carboxylic acid, D-or-L-1-oxazolidine-4-carboxylicacid (Kauer, U.S. Pat. No. (4,511,390) Serine S D-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met (O), D-Met (O), L-Cys, D-Cys Threonine TD-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met (O), D-Met (O), Val, D-ValTyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu,D-Leu, Ile, D-Ile, Met, D-Met, AdaA, AdaG

Control: As used herein, refers to the art-understood meaning of a“control” being a standard against which results are compared.Typically, controls are used to augment integrity in experiments byisolating variables in order to make a conclusion about such variables.In some embodiments, a control is a reaction or assay that is performedsimultaneously with a test reaction or assay to provide a comparator. A“control” also includes a “control animal.” A “control animal” may havea modification as described herein, a modification that is different asdescribed herein, or no modification (i.e., a wild-type animal). In oneexperiment, a “test” (i.e., a variable being tested) is applied. In asecond experiment, the “control,” the variable being tested is notapplied. In some embodiments, a control is a historical control (i.e.,of a test or assay performed previously, or an amount or result that ispreviously known). In some embodiments, a control is or comprises aprinted or otherwise saved record. A control may be a positive controlor a negative control.

Determining, measuring, evaluating, assessing, assaying and analyzing:Determining, measuring, evaluating, assessing, assaying and analyzingare used interchangeably herein to refer to any form of measurement, andinclude determining if an element is present or not. These terms includeboth quantitative and/or qualitative determinations. Assaying may berelative or absolute. “Assaying for the presence of” can be determiningthe amount of something present and/or determining whether or not it ispresent or absent.

Dosage form: Those skilled in the art will appreciate that the term“dosage form” may be used to refer to a physically discrete unit of anagent (e.g., a therapeutic agent) for administration to a subject.Typically, each such unit contains a predetermined quantity of agent. Insome embodiments, such quantity is a unit dosage amount (or a wholefraction thereof) appropriate for administration in accordance with adosing regimen that has been determined to correlate with a desired orbeneficial outcome when administered to a relevant population (i.e.,with a therapeutic dosing regimen). Those of ordinary skill in the artappreciate that the total amount of a therapeutic composition or agentadministered to a particular subject is determined by one or moreattending physicians and may involve administration of multiple dosageforms.

Dosing regimen: Those skilled in the art will appreciate that the term“dosing regimen” may be used to refer to a set of unit doses (typicallymore than one) that are administered individually to a subject,typically separated by periods of time. In some embodiments, a givenagent has a recommended dosing regimen, which may involve one or moredoses. In some embodiments, a dosing regimen comprises a plurality ofdoses each of which is separated in time from other doses. In someembodiments, individual doses are separated from one another by a timeperiod of the same length; in some embodiments, a dosing regimencomprises a plurality of doses and at least two different time periodsseparating individual doses. In some embodiments, all doses within adosing regimen are of the same unit dose amount. In some embodiments,different doses within a dosing regimen are of different amounts. Insome embodiments, a dosing regimen comprises a first dose in a firstdose amount, followed by one or more additional doses in a second doseamount different from the first dose amount. In some embodiments, adosing regimen comprises a first dose in a first dose amount, followedby one or more additional doses in a second dose amount same as thefirst dose amount. In some embodiments, a dosing regimen is correlatedwith a desired or beneficial outcome when administered across a relevantpopulation.

Engineered: In general, the term “engineered” refers to the aspect ofhaving been manipulated by the hand of man. For example, a cell ororganism is considered to be “engineered” if it has been manipulated sothat its genetic information is altered (e.g., new genetic material notpreviously present has been introduced, for example by transformation,mating, somatic hybridization, transfection, transduction, or othermechanism, or previously present genetic material is altered or removed,for example by substitution or deletion mutation, or by matingprotocols). As is common practice and is understood by those in the art,progeny of an engineered polynucleotide or cell are typically stillreferred to as “engineered” even though the actual manipulation wasperformed on a prior entity.

Excipient: As used herein, refers to an inactive (e.g., non-therapeutic)agent that may be included in a pharmaceutical composition, for exampleto provide or contribute to a desired consistency or stabilizing effect.In some embodiments, suitable pharmaceutical excipients may include, forexample, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized. A biological molecule may havetwo functions (i.e., bifunctional) or many functions (i.e.,multifunctional).

Gene: As used herein, refers to a DNA sequence in a chromosome thatcodes for a product (e.g., an RNA product and/or a polypeptide product).In some embodiments, a gene includes coding sequence (i.e., sequencethat encodes a particular product). In some embodiments, a gene includesnon-coding sequence. In some particular embodiments, a gene may includeboth coding (e.g., exonic) and non-coding (e.g., intronic) sequence. Insome embodiments, a gene may include one or more regulatory sequences(e.g., promoters, enhancers, etc.) and/or intron sequences that, forexample, may control or impact one or more aspects of gene expression(e.g., cell-type-specific expression, inducible expression, etc.). Forthe purpose of clarity, we note that, as used in the present disclosure,the term “gene” generally refers to a portion of a nucleic acid thatencodes a polypeptide or fragment thereof, the term may optionallyencompass regulatory sequences, as will be clear from context to thoseof ordinary skill in the art. This definition is not intended to excludeapplication of the term “gene” to non-protein-coding expression unitsbut rather to clarify that, in most cases, the term as used in thisdocument refers to a polypeptide-coding nucleic acid.

Improve, increase, enhance, inhibit or reduce: As used herein, the terms“improve,” “increase,” “enhance,” “inhibit,” “reduce,” or grammaticalequivalents thereof, indicate values that are relative to a baseline orother reference measurement. In some embodiments, a value isstatistically significantly difference that a baseline or otherreference measurement. In some embodiments, an appropriate referencemeasurement may be or comprise a measurement in a particular system(e.g., in a single individual) under otherwise comparable conditionsabsent presence of (e.g., prior to and/or after) a particular agent ortreatment, or in presence of an appropriate comparable reference agent.In some embodiments, an appropriate reference measurement may be orcomprise a measurement in comparable system known or expected to respondin a particular way, in presence of the relevant agent or treatment. Insome embodiments, an appropriate reference is a negative reference; insome embodiments, an appropriate reference is a positive reference.

Isolated: As used herein, refers to a substance and/or entity that hasbeen (1) separated from at least some of the components with which itwas associated when initially produced (whether in nature and/or in anexperimental setting), and/or (2) designed, produced, prepared, and/ormanufactured by the hand of man. In some embodiments, an isolatedsubstance or entity may be enriched; in some embodiments, an isolatedsubstance or entity may be pure. In some embodiments, isolatedsubstances and/or entities may be separated from about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more than about 99% of the othercomponents with which they were initially associated. In someembodiments, isolated agents are about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% pure. As used herein, asubstance is “pure” if it is substantially free of other components. Insome embodiments, as will be understood by those skilled in the art, asubstance may still be considered “enriched”, “isolated” or even “pure”,after having been combined with certain other components such as, forexample, one or more carriers or excipients (e.g., buffer, solvent,water, etc.); in such embodiments, percent isolation or purity of thesubstance is calculated without including such carriers or excipients.Those skilled in the art are aware of a variety of technologies forisolating (e.g., enriching or purifying) substances or agents (e.g.,using one or more of fractionation, extraction, precipitation, or otherseparation).

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” refers to a composition in which an active agent isformulated together with one or more pharmaceutically acceptablecarriers. In some embodiments, the active agent is present in unit doseamount appropriate for administration in a therapeutic regimen thatshows a statistically significant probability of achieving apredetermined therapeutic effect when administered to a relevantpopulation. In some embodiments, a pharmaceutical composition may bespecially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue, capsules, powders, etc. In some embodiments, an active agent maybe or comprise a cell or population of cells (e.g., a culture, forexample of an EES microbe); in some embodiments, an active agent may beor comprise an extract or component of a cell or population (e.g.,culture) of cells. In some embodiments, an active agent may be orcomprise an isolated, purified, or pure compound. In some embodiments,an active agent may have been synthesized in vitro (e.g., via chemicaland/or enzymatic synthesis). In some embodiments, an active agent may beor comprise a natural product (whether isolated from its natural sourceor synthesized in vitro).

Pharmaceutically acceptable: As used herein, the term “pharmaceuticallyacceptable” which, for example, may be used in reference to a carrier,diluent, or excipient used to formulate a pharmaceutical composition asdisclosed herein, means that the carrier, diluent, or excipient iscompatible with the other ingredients of the composition and notdeleterious to the recipient thereof.

Pharmaceutically acceptable carrier: As used herein, the term“pharmaceutically acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be is “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

Prevention: The term “prevention”, as used herein, refers to a delay ofonset, and/or reduction in frequency and/or severity of one or moresymptoms of a particular disease, disorder or condition. In someembodiments, prevention is assessed on a population basis such that anagent is considered to “prevent” a particular disease, disorder orcondition if a statistically significant decrease in the development,frequency, and/or intensity of one or more symptoms of the disease,disorder or condition is observed in a population susceptible to thedisease, disorder, or condition. In some embodiments, prevention may beconsidered complete, for example, when onset of a disease, disorder orcondition has been delayed for a predefined period of time.

Reference: As used herein describes a standard or control relative towhich a comparison is performed. For example, in some embodiments, anagent, animal, individual, population, sample, sequence or value ofinterest is compared with a reference or control agent, animal,individual, population, sample, sequence or value. In some embodiments,a reference or control is tested and/or determined substantiallysimultaneously with the testing or determination of interest. In someembodiments, a reference or control is a historical reference orcontrol, optionally embodied in a tangible medium. Typically, as wouldbe understood by those skilled in the art, a reference or control isdetermined or characterized under comparable conditions or circumstancesto those under assessment. Those skilled in the art will appreciate whensufficient similarities are present to justify reliance on and/orcomparison to a particular possible reference or control. In someembodiments, a reference is a negative control reference; in someembodiments, a reference is a positive control reference.

Risk: As will be understood from context, “risk” of a disease, disorder,and/or condition refers to a likelihood that a particular individualwill develop the disease, disorder, and/or condition. In someembodiments, risk is expressed as a percentage. In some embodiments,risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, or up to 100%. In some embodiments risk is expressed as a riskrelative to a risk associated with a reference sample or group ofreference samples. In some embodiments, a reference sample or group ofreference samples have a known risk of a disease, disorder, conditionand/or event. In some embodiments a reference sample or group ofreference samples are from individuals comparable to a particularindividual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more.

Sample: As used herein, the term “sample” typically refers to an aliquotof material obtained or derived from a source of interest. In someembodiments, a source of interest is a biological or environmentalsource. In some embodiments, a source of interest may be or comprise acell or an organism, such as a microbe, a plant, or an animal (e.g., ahuman). In some embodiments, a source of interest is or comprisesbiological tissue or fluid. In some embodiments, a biological tissue orfluid may be or comprise amniotic fluid, aqueous humor, ascites, bile,bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle,chime, ejaculate, endolymph, exudate, feces, gastric acid, gastricjuice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid,pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum,synovial fluid, sweat, tears, urine, vaginal secretions, vitreoushumour, vomit, and/or combinations or component(s) thereof. In someembodiments, a biological fluid may be or comprise an intracellularfluid, an extracellular fluid, an intravascular fluid (blood plasma), aninterstitial fluid, a lymphatic fluid, and/or a transcellular fluid. Insome embodiments, a biological fluid may be or comprise a plant exudate.In some embodiments, a biological tissue or sample may be obtained, forexample, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab(e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washingor lavage (e.g., bronchioalveolar, ductal, nasal, ocular, oral, uterine,vaginal, or other washing or lavage). In some embodiments, a biologicalsample is or comprises cells obtained from an individual. In someembodiments, a sample is a “primary sample” obtained directly from asource of interest by any appropriate means. In some embodiments, aswill be clear from context, the term “sample” refers to a preparationthat is obtained by processing (e.g., by removing one or more componentsof and/or by adding one or more agents to) a primary sample. Forexample, filtering using a semi-permeable membrane. Such a “processedsample” may comprise, for example nucleic acids or proteins extractedfrom a sample or obtained by subjecting a primary sample to one or moretechniques such as amplification or reverse transcription of nucleicacid, isolation and/or purification of certain components, etc.

Small molecule: As used herein, the term “small molecule” refers tosmall organic or inorganic molecules of molecular weight below about3,000 Daltons. In general, small molecules may have a molecular weightof less than 3,000 Daltons (Da). Small molecules can be, e.g., from atleast about 100 Da to about 3,000 Da (e.g., between about 100 to about3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about100 to about 500 Da, about 200 to about 1500, about 500 to about 1000,about 300 to about 1000 Da, or about 100 to about 250 Da).

Subject: As used herein, the term “subject” refers to an individual towhich a provided treatment is administered. In some embodiments, asubject is animal. In some embodiments, a subject is a mammal, e.g., amammal that experiences or is susceptible to a disease, disorder, orcondition as described herein. In some embodiments, an animal is avertebrate, e.g., a mammal, such as a non-human primate, (particularly ahigher primate), a sheep, a dog, a rodent (e.g. a mouse or rat), aguinea pig, a goat, a pig, a cat, a rabbit, or a cow. In someembodiments, an animal is a non-mammal animal, such as a chicken, anamphibian, a reptile, or an invertebrate model C. elegans. In someembodiments, a subject is a human. In some embodiments, a patient issuffering from or susceptible to one or more diseases, disorders orconditions as described herein. In some embodiments, a patient displaysone or more symptoms of a one or more diseases, disorders or conditionsas described herein. In some embodiments, a patient has been diagnosedwith one or more diseases, disorders or conditions as described herein.In some embodiments, the subject is receiving or has received certaintherapy to diagnose and/or to treat a disease, disorder, or condition.In another embodiment, the subject is an experimental animal or animalsubstitute as a disease model.

Substantially: As used herein, refers to the qualitative condition ofexhibiting total or near-total extent or degree of a characteristic orproperty of interest. One of ordinary skill in the biological arts willunderstand that biological and chemical phenomena rarely, if ever, go tocompletion and/or proceed to completeness or achieve or avoid anabsolute result. The term “substantially” is therefore used herein tocapture the potential lack of completeness inherent in many biologicaland chemical phenomena.

Therapeutic regimen: A “therapeutic regimen”, as that term is usedherein, refers to a dosing regimen whose administration across arelevant population may be correlated with a desired or beneficialtherapeutic outcome.

Therapeutically effective amount: As used herein, is meant an amountthat produces the desired effect for which it is administered. In someembodiments, the term refers to an amount that is sufficient, whenadministered to a population suffering from or susceptible to a disease,disorder, and/or condition in accordance with a therapeutic dosingregimen, to treat the disease, disorder, and/or condition. In someembodiments, a therapeutically effective amount is one that reduces theincidence and/or severity of, and/or delays onset of, one or moresymptoms of the disease, disorder, and/or condition. Those of ordinaryskill in the art will appreciate that the term “therapeuticallyeffective amount” does not in fact require successful treatment beachieved in a particular individual. Rather, a therapeutically effectiveamount may be that amount that provides a particular desiredpharmacological response in a significant number of subjects whenadministered to patients in need of such treatment. In some embodiments,reference to a therapeutically effective amount may be a reference to anamount as measured in one or more specific tissues (e.g., a tissueaffected by the disease, disorder or condition) or fluids (e.g., blood,saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill inthe art will appreciate that, in some embodiments, a therapeuticallyeffective amount of a particular agent or therapy may be formulatedand/or administered in a single dose. In some embodiments, atherapeutically effective agent may be formulated and/or administered ina plurality of doses, for example, as part of a dosing regimen.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a therapy that partially orcompletely alleviates, ameliorates, relives, inhibits, delays onset of,reduces severity of, and/or reduces incidence of one or more symptoms,features, and/or causes of a particular disease, disorder, and/orcondition. In some embodiments, such treatment may be of a subject whodoes not exhibit signs of the relevant disease, disorder and/orcondition and/or of a subject who exhibits only early signs of thedisease, disorder, and/or condition. Alternatively, or additionally,such treatment may be of a subject who exhibits one or more establishedsigns of the relevant disease, disorder and/or condition. In someembodiments, treatment may be of a subject who has been diagnosed assuffering from the relevant disease, disorder, and/or condition. In someembodiments, treatment may be of a subject known to have one or moresusceptibility factors that are statistically correlated with increasedrisk of development of the relevant disease, disorder, and/or condition.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows data evidencing that APOE4 protein enhances the Aβ-inducedparalysis phenotype in C. elegans. C. elegans animals having theindicated genotypes were administered an E. coli OP50 standardlaboratory strain. Animals were monitored from day 1 adulthood everyalternative day until all were paralyzed. For each assay, at least 20animals were recorded. Data from three independent trials are presentedin a plot. For each data point, mean±s.d is presented in the graph.

FIG. 2 shows data evidencing that expression of Aβ₃₋₄₂ conjugated togreen fluorescent protein (GFP) and human ssAPOE4 significantlyincreased the number of aggregates. C. elegans animals having theindicated genotypes were administered an E. coli OP50 standardlaboratory strain. GFP aggregates in the anterior region of the animalwere counted when the animals reached adulthood. For each assay, atleast 17 animals were recorded. For each data point, mean±s.d ispresented in a bar graph. Compared to animals expressing Aβ3-42, animalsexpressing Aβ3-42 and ssApoE4 had significantly increased GFP aggregatesas analyzed using Student's t-test, P<0.0001.

FIG. 3 shows data evidencing that UbV-GFP is stabilized in animalsexpressing both human ssAPOE4 and human tau352(PHP). C. elegans animalshaving the indicated genotypes were administered with the E. coli OP50standard laboratory strain. The number of animals expressing GFP in thegut were counted when the animals reached adulthood. For each assay, atleast 30 animals were recorded. Data from three independent trials arepresented in a bar graph. For each data point, mean±s.d is presented inthe graph. Compared to animals that expressed tau352(PHP), animalsexpressing ssAPOE4 and tau352(PHP) had significantly increased UbV-GFPexpression as analyzed using Student's t-test, P<0.0001.

FIG. 4 includes data evidencing that certain microbial strains of amicrobiome increase Aβ mediated paralysis. C. elegans animals having theindicated genotypes were administered with either E. coli OP50 standardlaboratory strain or individual microbiome strains. Number of paralyzedanimals were recorded on day 4 of adulthood. Data from three independenttrials are presented in a bar graph. For each data point, mean±s.d ispresented in the graph. Table 1 includes the raw data with number ofanimals analyzed for each condition.

FIG. 5 includes data evidencing certain microbial strains of amicrobiome modulate Aβ3-42::GFP aggregation. C. elegans animals havingthe indicated genotypes were administered with either E. coli op50standard laboratory strain or individual microbiome strains. The numberof GFP aggregates in the anterior region of the animal were counted whenthe animals reached adulthood. GFP aggregates in three animals werecounted for each condition. For each data point, mean±s.d is presentedin the graph.

FIG. 6 includes data showing that certain microbial strains altered ATPproduction. Neuro2A cells were either mock-treated or treated with 10⁸CFU of each bacterium or a combination of all bacteria (CT10) for 16hours. 2 μM of Human Amyloid β₁₋₄₂ was added to all the wells except forthe untreated wells. Cells were incubated for 24 hours and ATP levelswere measured and normalized. The percent ATP was calculated accordingto the following formula: % ATP=[(sample normalized luminescencevalue/control normalized luminescence) *100].

FIG. 7 includes a schematic of the exemplary HIF pathways.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Provided herein are transgenic C. elegans disease models and methods ofusing C. elegans disease models to rapidly identify, assess, orcharacterize one or more microbial strains from a mammalian microbiomeas affecting (e.g., enhancing or minimizing) a phenotype associated witha mammalian disease or condition. In some embodiments, identification,assessment, or characterization can occur with or without chemicalentities and/or biologics (e.g., antibodies).

C. elegans is a bacteriovorus nematode that is fed on E. coli diet inthe laboratory. C. elegans can provide reliable, effective, andefficient genotypic and phenotypic models for a number of mammaliandiseases and conditions, including various human diseases andconditions, such as aging, diabetes, neurodegenerative disorders,metabolic diseases, and cancer. The present disclosure provides theinsight that C. elegans models of mammalian diseases and conditions canbe used to rapidly screen human microbiome for microbial strains thatare affecting such mammalian diseases and conditions. It is improbableto conduct such studies in standard mammalian cell culture assays oranimal models. For example, it would be economically unviable,time-consuming, and laborious to screen millions of microbial speciesand/or strains individually, or even in combinations using, e.g., mousemodels of human diseases and conditions. While C. elegans may notinclude some of the complexities of mammalian systems, C. elegans modelsshare features with mammalian systems, have quick response times, andare less expensive to produce and maintain than mammalian model systems.Accordingly, C. elegans models of mammalian diseases and conditionsprovide a powerful front line for examining the impacts microbialstrains of mammalian biomes have on mammalian diseases and conditionsand present a useful tool to prioritize lead microbial strains thatimpact specific and sensitive conserved therapeutic targets.

Provided herein are methods of using C. elegans as a tool for rapidlyscreening human microbiome for disease modifiers. Microbial strains ofsamples from a healthy patient or a patient having a disease orcondition can be cultured using standard microbiological techniques.These microbial strains can be fed to a transgenic C. elegans straincarrying a mammalian (e.g., human) disease marker, a mammalian (e.g.,human) disease gene mutation, or a combination thereof. Microbialstrains can be fed to a transgenic C. elegans strain either individuallyor in combination. In some cases, an individual microbial strain or acombination of microbial strains can be fed to transgenic C. elegansstrains that have a mammalian (e.g., human) disease marker, a mammalian(e.g., human) disease gene mutation, or a combination thereof incombination with chemical entities (e.g., small molecules, e.g., drugs)or biologics (e.g., monoclonal antibodies).

The present disclosure recognizes that multiple outcomes can arise fromfeeding an individual microbial strain or a combination of microbialstrains can be fed to transgenic C. elegans strains that have amammalian (e.g., human) disease marker, a mammalian (e.g., human)disease gene mutation, or a combination thereof in combination withchemical entities (e.g., small molecules, e.g., drugs) or biologics(e.g., monoclonal antibodies). In a first scenario, methods describedhere could be used to identify, define, assess, and/or detect individualmicrobial strains or combinations of microbial strains from a mammalianmicrobiome that increase the severity of a mammalian disease orcondition phenotype in C. elegans. In a second scenario, methodsdescribed here could be used to identify, define, assess, and/or detectindividual microbial strains or combinations of microbial strains from amammalian microbiome that decrease the severity of a mammalian diseaseor condition phenotype in C. elegans. In a third scenario, methodsdescribed here could be used to identify, define, assess, and/or detectindividual microbial strains or combinations of microbial strains from amammalian microbiome that have no effect of a mammalian disease orcondition phenotype in C. elegans. The present disclosure recognizesthat each of these outcomes provides valuable information.

For example, individual microbial strains or combinations of microbialstrains from a mammalian microbiome that increase the severity of amammalian disease or condition phenotype in C. elegans can be potentialearly diagnostic biomarkers for a mammalian disease or condition. Insome embodiments, such individual microbial strains or combinations ofmicrobial strains are correlated with a high occurrence or increasedseverity of the disease or condition in mammals (e.g., human). If such acorrelation has not been previously found, transgenic C. elegans andmethods of using transgenic C. elegans described herein could be used torapidly screen and/or assess one or more microbial strains in amammalian microbiome using genetic screening or chemical extraction orgenomic data mining methods to identify the potential “toxic”metabolites or components of microbiome involved in an increase indisease severity or incidence. These identified microbial strains and/orcomponents of a mammalian microbiome could be also used for developingdiagnostics. Also, identification and/or characterization of “toxic”microbial strains or microbiome components could be used for developingmodulators or therapeutics that could target microbial strains,microbiome components or biosynthetic pathways that produce them.

In addition, the present disclosure recognizes that individual microbialstrains or combinations of microbial strains from a mammalian microbiomethat decrease the severity of a mammalian disease or condition phenotypein C. elegans can correlate with disease severity in human patients. Insome embodiments, such individual microbial strains or combinations ofmicrobial strains are correlated with a low occurrence or decreasedseverity of the disease or condition in mammals (e.g., human). If such acorrelation has not been previously found, transgenic C. elegans andmethods of using transgenic C. elegans described herein could be used torapidly screen and/or assess one or more microbial strains in amammalian microbiome using genetic screening or chemical extraction orgenomic data mining methods to identify the potential “beneficial”microbial strains, microbiome components, or metabolites involved in andecrease in disease severity or incidence. These identified microbialstrains and/or components of a mammalian microbiome could be also usedfor developing diagnostics. Also, identification and/or characterizationof “beneficial” microbial strains or microbiome components could be usedas modulators or therapeutics for disease.

In some case, individual microbial strains or combinations of microbialstrains from a mammalian microbiome can be also fed in combination withchemical entities (e.g., small molecules, e.g., drugs) or biologics(e.g., monoclonal antibodies), or combinations thereof to a transgenicC. elegans strain to identify potential biological or signaling orcellular target genes or pathways. Biological or signaling or cellulartarget genes or pathways can include, but are not limited to,inflammation, insulin receptor, cell death, mitochondria, endoplasmicreticulum, proteasome, lipogenesis, and detoxification.

In some cases, individual microbial strains or combinations of microbialstrains from a mammalian microbiome can be fed in combination withchemical entities (e.g., small molecules, e.g., drugs) or biologics(e.g., monoclonal antibodies), or combinations thereof to C. elegansstrain to identify a set of signaling or target genes or pathways (e.g.,a comprehensive set) that could modulate or optimize functions of aparticular subcellular organelle relevant for a particular disease. Forexample, there are multiple pathways or targets that could be modulatedto achieve optimal mitochondrial function, which is a relevant andimportant target in several neurodegenerative diseases, includingAlzheimer's disease (AD). Biological targets for improving mitochondrialfunction include: biogenesis, bioenergetics, hormesis, and/or repair.Using transgenic C. elegans disease models and/or methods of using C.elegans disease models described herein, it is possible to identifyindividual or combinatorial microbiome species and/or combination withchemical entities (e.g., small molecules, e.g., drugs) or biologics(e.g., monoclonal antibodies), or combinations thereof that could modifyor improve or alter either mitochondrial biogenesis, bioenergetics,hormesis or repair or combinations thereof. Thus, it is possible toidentify and combine individual microbial strains or combinations ofmicrobial strains from a mammalian microbiome and/or chemical entities(e.g., small molecules, e.g., drugs) or biologics (e.g., monoclonalantibodies), or combinations thereof that able to target multiplepathways to, e.g., achieve optimal mitochondrial function.

C. elegans

The free-living nematode C. elegans has been used extensively as a modelsystem. C. elegans are inexpensive to cultivate, easy to physicallymanipulate, and has a multitude of genetic and molecular tools availablefor study. C. elegans are simple multicellular organisms: adults containapproximately 1,000 somatic cells yet have a variety of tissue typessuch as muscles, nerves, and intestinal cells. C. elegans have a shortgeneration time, which allows for rapid experimentation. C. elegansgenerally progress from egg to larva to fertile adult in 3 days at roomtemperature. A single adult C. elegans can have between 300 and 1,000progenies, which allows for a significant number of animals to be usedand then quickly replenished in a relatively short amount of time. Dueto the sexual dimorphism, C. elegans are useful for genetics.Self-fertilizing hermaphrodites can be maintained as homozygousmutations without the need for mating and males can be used for geneticcrosses. C. elegans are transparent at every stage of their life cycle,which provides the ability to see inside the organism. This permits theobservation of cellular events. It also permits the use ofphosphorescent, luminescent, and fluorescent reporters. Manipulation ofprotein expression in C. elegans can also be performed usingRNA-mediated interference (RNAi), which can allow for rapid assessmentof gene function. Another advantage of using C. elegans a model systemis the ability to freeze and recover the animals, thereby allowinglong-term storage.

C. elegans can be genetically modified using a number of techniques togenerate transgenic C. elegans strains. The sexual dimorphism of C.elegans allows for genetic manipulations to be performed with relativeease and according to know procedures. For example, if a strain needs tobe propagated, single hermaphrodites can be used to self-fertilize andgenerate a population of offspring. Even if a mutation renders an animalunable to mate, it remains possible for a hermaphrodite to produceprogeny. Another aspect of C. elegans reproduction that makes C. elegansan effective genetic tool is the animal's ability to cross males withhermaphrodites. For example, mating experiments allow genetic markerssuch as mutations causing visible phenotypes to be placed together in asingle organism along with an unknown mutation in order to facilitatemapping of that mutation. Hermaphrodites make only a limited number ofsperm and can typically have approximately 300 self progeny. Matingincreases the number of offspring produced by a single hermaphrodite toapproximately 1,000 due to the addition of the male-produced sperm. Therelatively large number of progeny coupled with the short life span ofC. elegans allows for rapid and inexpensive analyses to be performed onthe animals.

In addition to genetic modifications via reproduction, C. elegans can begenetically modified via injection of transgenes. Microinjection is aneffective method for creating transgenic animals and for introducingvarious types of molecules directly to cells. For DNA transformation,one approach is to inject DNA into a distal arm of a C. elegans gonad. Adistal germline of C. elegans contains a central core of cytoplasm thatis shared by many germ cell nuclei. Therefore, DNA injected into adistal arm of a C. elegans gonad can be delivered to many progeny.Microinjection directly into oocyte nuclei can induce chromosomalintegration of transgenes, but this technique can be more difficult toperform. C. elegans can also incorporate genetic material that is fed tothem.

C. elegans are relatively simple to culture. C. elegans can becultivated in either liquid culture or on the Nematode Growth Medium(NGM) agar plates in the presence of bacteria. It is possible to growthe animals in a chemically defined medium without the addition ofbacteria, which can be useful because the components of a medium can bealtered in order to study the nutrient or other chemical requirements ofthe animals. In some embodiments, C. elegans are grown on the agarplates. C. elegans can be grown on Nematode Growth Medium (NGM) agarplates. Bacteria can be spread on the NGM plates as a food source forthe animals. For example, OP50, a leaky E. coli uracil auxotroph can beused. OP50 will grow slowly and provide nutrients for the animalswithout overgrowing them. Once the animals have eaten all of the food ona plate they will burrow into the agar and can be maintained on the“starved” plate for weeks at a time in a 15° C. incubator. The animalscan be transferred to an agar plate with fresh bacteria by eithercutting and moving a small block of agar from the starved plate with asterile instrument such as a micropipette tip, or washing the worms offthe surface of the plate with sterile water, or by picking one or moreindividuals onto a fresh plate, which will cause the C. elegans toreemerge. At any time, C. elegans can be cryogenically preserved. C.elegans prefer to grow between 15° C. and 25° C., but the temperaturecan vary depending on the strain of C. elegans and conditions beingtested. In some embodiments, a C. elegans culture can be cultured at atemperature of at least 5° C., at least 10° C., at least 15° C., atleast 20° C., at least 25° C., at least 30° C., at least 35° C., or atleast 40° C. In some embodiments, a C. elegans culture can be culturedat a temperature of at most 65° C., at most 60° C., at most 55° C., atmost 50° C., at most 55° C., at most 40° C., at most 35° C., at most 30°C., at most 25° C., or at most 20° C. Standard protocols for C. elegansmanipulation and culture are known, e.g., as described by Stiernagle T.Maintenance of C. elegans. Wormbook, ed. The C. elegans ResearchCommunity, WormBook. (Feb. 11, 2006), which is incorporated herein byreference.

Microbial Preparation(s) and/or Component(s)

The present disclosure provides systems and methods for assessing,characterizing, and identifying one or more microbial strains of amicrobiome. Such systems and methods can be useful for assessing,characterizing, and identifying one or more microbial strains thataffect the health of humans, livestock, and/or pets. In someembodiments, assessing, characterizing, and identifying one or moremicrobial strains from a microbiome of a snake, lizard, fish, or bird.In some embodiments, assessing, characterizing, and identifying one ormore microbial strains from a mammalian microbiome. A mammalianmicrobiome can be a canine, a feline, an equine, a bovine, an ovine, acaprine, or a porcine microbiome. Generally, a microbiome used in asystem or method described herein will correspond with the disease orcondition modeled by a transgenic C. elegans used in the system ormethod. For example, if a transgenic C. elegans models a human disease,a human microbiome will be assessed, characterized, or identified.

A microbiome can be isolated from any system or tissue of an organismthat supports microbial growth. For example, a microbiome can be acutaneous microbiome, an oral microbiome, a nasal microbiome, agastrointestinal microbiome, a brain microbiome, a pulmonary microbiome,or a urogenital microbiome. A list of exemplary microbial strains foundin a gastrointestinal microbiome is included below in TABLE 8. A personskilled in the art would understand that a microbiome sample can beobtained by various ways known in the art. For example, a cutaneous,oral, nasal, pulmonary, or urogenital microbiome sample could beobtained using a swab or tissue scrapping. In some embodiments, agastrointestinal microbiome could be sampled from feces. A cutaneousmicrobiome, an oral microbiome, a nasal microbiome, a gastrointestinalmicrobiome, a brain microbiome, a pulmonary microbiome, or a urogenitalmicrobiome sample could be obtained via a biopsy.

In some embodiments, a microbiome is a microbiome of a healthyindividual or an individual who does not suffer from or is not at riskof developing a particular disease or disorder. In some embodiments, amicrobiome is a microbiome of an individual that suffers from or is atrisk of developing a particular disease or disorder. In someembodiments, a microbiome is a microbiome of an individual who is knownto suffer from a particular disease or disorder. In some embodiments, ahuman microbiome is a microbiome of a human with an unknown risk for oneor more diseases or conditions.

In some embodiments, a microbiome is a reference microbiome. A referencemicrobiome can be a microbiome of a healthy individual or an individualwho does not suffer from or is not at risk of developing a particulardisease or disorder. In some instances, a reference microbiome may befrom the same individual as a microbiome to be assessed orcharacterized, but was obtained at a different time. In some instances,a reference microbiome may be from the same individual as a microbiometo be assessed or characterized, but was obtained from a differentsystem or tissue.

In some embodiments, an individual microbial strain or a combination ofmicrobial strains may be assessed, characterized, or identified in adifferent relative amount than such strain or strains are found in amicrobiome. For example, a single strain may be assessed, characterized,or identified using transgenic C. elegans or methods using transgenic C.elegans described herein, even though it is naturally present in amicrobiome with other microbial strains. As another example, twomicrobial strains may be assessed, characterized, or identified togethertransgenic C. elegans or methods using transgenic C. elegans describedherein, even though they are naturally present in a microbiome withadditional microbial strains.

An extract, component, or compound of a microbial strain may also beassessed, characterized, or identified using transgenic C. elegans ormethods using transgenic C. elegans described herein. In some cases, anextract, component, or compound of a microbial strain that has beendetermined to affect a transgenic C. elegans model of a disease orcondition may be assessed, characterized, or identified. Assessing,characterizing or identifying an extract, component, or compound of amicrobial strain that affect a transgenic C. elegans model of a diseaseor condition may provide additional information about potentialbiomarkers, targets, or protective agents in a microbiome.

A variety of technologies are known in the art that can be used toprepare extracts of microbial strains, and/or to isolate extracts,components, or compounds therefrom, or to process (e.g., to isolateand/or purify one or more components or compounds from). To give but afew examples, such technologies may include, for example, one or more oforganic extraction, vacuum concentration, chromatography, and so on.

Assessing Biological Impact

The present disclosure provides the insight that C. elegans can be usedto identify, characterize, or assess microbial strain(s) of a mammalianmicrobiome by contacting the microbial strain(s) (e.g., feeding themicrobial strain(s) to, administering to) transgenic C. elegans thatmodel a mammalian disease or condition. To determine whether a microbialstrain or combination of microbial strains affects a transgenic C.elegans that model a mammalian disease or condition, parameters of thetransgenic C. elegans can be observed, measured, or assessed indifferent samples that have been contacted with the microbial strain orcombination of microbial strains. Various parameters of transgenic C.elegans can be observed, measured, or assessed to determine whether amicrobial strain or combination of microbial strains affects atransgenic C. elegans that model a mammalian disease or condition. Asjust a few examples, transgenic C. elegans behaviors (e.g., mating,feeding, food aversion, or locomotion), genetic mutations (e.g., thepresence of SNPs, deletions, additions, inversions, or repeats in DNA),transcript levels, protein levels, metabolite levels, lipid levels,carbohydrate levels, protein (e.g., enzyme) activity levels can beobserved, measured, or assessed to determine whether a microbial strainor combination of microbial strains affects a transgenic C. elegans thatmodel a mammalian disease or condition.

In some embodiments, methods described herein utilize a first sample anda second sample. In some embodiments, a first sample is a referencesample. In some embodiments, a reference sample can be a culture oftransgenic C. elegans contacted with (e.g., administered or fed), e.g.,OP50. In some embodiments, a reference sample can be a culture oftransgenic C. elegans contacted with (e.g., administered or fed) amicrobial strain or combination of microbial strains from a microbiomeof a healthy individual. In some embodiments, a reference sample can bea culture of transgenic C. elegans contacted with (e.g., administered orfed) a microbial strain or combination of microbial strains from amicrobiome of an individual obtained at a first time point.

In some embodiments, a second sample can be a test sample. In someembodiments, a test sample can be a culture of transgenic C. eleganscontacted with (e.g., administered or fed) an individual microbialstrain or a combination of microbial strains from a mammalianmicrobiome, e.g., a human microbiome. In some instances, a humanmicrobiome is a microbiome of a human suffering from or at risk of adisease or condition. In some instances, a human microbiome is amicrobiome of a human with an unknown risk for one or more diseases orconditions. In some embodiments, a test sample can be a culture oftransgenic C. elegans contacted with (e.g., administered or fed) amicrobial strain or combination of microbial strains from a microbiomeof an individual obtained at a second time point.

In some embodiments, methods described herein comprise comparing one ormore parameters obtained from a test sample with one or more parametersobtained from a reference sample. In some embodiments, by comparing oneor more parameters obtained from a test sample with one or moreparameters obtained from a reference sample, it can be determined thatan individual microbial strain or a combination of microbial strainsfrom a microbiome increase the severity or incidence of a disease orcondition phenotype modeled by the cultured transgenic C. elegans. Insome embodiments, by comparing one or more parameters obtained from atest sample with one or more parameters obtained from a referencesample, it can be determined that an individual microbial strain or acombination of microbial strains from a microbiome decrease the severityor incidence of a disease or condition phenotype modeled by the culturedtransgenic C. elegans. In some embodiments, by comparing one or moreparameters obtained from a test sample with one or more parametersobtained from a reference sample, it can be determined that anindividual microbial strain or a combination of microbial strains from amicrobiome have no effect on the severity or incidence of a disease orcondition phenotype modeled by the cultured transgenic C. elegans.

Transgenic C. elegans and methods using transgenic C. elegans providedherein can be useful in assessing, characterizing, or identifyingmicrobial strains of a microbiome that affect a mammalian disease orcondition. The present disclosure also provides the recognition thattransgenic C. elegans and methods using transgenic C. elegans providedherein can be used to define and/or characterize a microbial signatureassociated with a disease or condition. Further, the present disclosureprovides the recognition that transgenic C. elegans and methods usingtransgenic C. elegans provided herein can be used to define and/orcharacterize a microbial signature associated with one or more featuresof a disease or condition (e.g., severity, responsiveness to therapy,etc.). For example, if multiple microbial strains are determined to beassociated with an increased severity of a disease or disorder, e.g.,across multiple individuals, the microbial strains, as well as theirrelative amounts, could be used as a signature to identify individualswho are at risk of developing an increased severity of the disease ordisorder. As another example, if multiple microbial strains aredetermined to be associated with an increased severity of a disease ordisorder, e.g., in a single individual, at certain times (e.g., afterremoval from a treatment), the microbial strains, as well as theirrelative amounts, could be used as a signature to identify when thatindividual is at risk of developing an increased severity of the diseaseor disorder.

The present disclosure also provides the recognition that transgenic C.elegans and methods using transgenic C. elegans provided herein can beused to diagnose an individual with a disease or condition. In fact,using a microbial signature associated with a disease or conditiondetermined through the use of transgenic C. elegans and methods usingtransgenic C. elegans provided herein, an individual can be diagnosedearly and/or identified as an individual at risk.

The present disclosure also provides the recognition that transgenic C.elegans and methods using transgenic C. elegans provided herein can beused to monitor progression of a disease or condition in an individual.For example, if microbial strains determined to increase the severity ofa disease or condition decrease in relative amount within a microbiome,it may indicate that the disease or condition is being attenuated, e.g.,by treatment or immune response.

The present disclosure also provides the insight that transgenic C.elegans and methods using transgenic C. elegans provided herein can beused to tailor treatments (e.g., therapies, nutraceuticals, and/orprobiotics) to an individual patient. In some embodiments, transgenic C.elegans and methods using transgenic C. elegans provided herein canprovide “personalized” therapy. In some cases, microbial strains withinan individual can be assessed, characterized, or identified to determineif they have an effect on a disease or disorder. Based on the results,the individual can be treated with one or more microbial strains toadjust the microbial strains (and/or component or compound thereof) intheir microbiome. In some instances, this will affect the disease orcondition the individual is suffering from or at risk of developing. Forexample, if an individual is determined to have a relatively low amountof one or more microbial strains that have been determined to decreasethe severity of a disease or condition, administration of the one ormore microbial strains that have been determined to decrease theseverity of a disease or condition to the individual (or an extract,component, or compound thereof) may attenuate the severity of theindividual's disease or condition.

Pharmaceutical Compositions

Provided herein are compositions comprising individual microbial strainsor combinations of microbial strains. In some embodiments, a compositioncomprises individual microbial strains or combinations of microbialstrains from a mammalian microbiome, extracts thereof, and/or componentsthereof, which have been assessed, identified, characterized or assayedusing transgenic C. elegans or methods as described herein. In someembodiments, a composition provided herein comprises two or more, threeor more, four or more, five or more, six or more, seven or more, eightor more, nine or more, or ten or more microbial strains from a mammalianmicrobiome, extracts thereof, and/or components thereof, which have beenassessed, identified, characterized or assayed using transgenic C.elegans or methods as described herein.

In some embodiments, a composition provided herein comprises two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, or ten or more microbial strainslisted in TABLE 8 below.

In some embodiments, a composition provided herein comprisesGluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcussp., L. plantarum, Clostridium butyricum, Paenibacillus sp., Veillonellasp., Bifidobacterium, Bacillus subtilis, Acidaminococcus sp., or acombination thereof. In some embodiments, a combination comprises atleast two of, at least three of, at least four of, at least five of, atleast six of, at least seven of, at least eight of, at least nine of, orall of Gluconacetobacter hansenii, Terrisporobacter glycolicus,Coprococcus sp., L. plantarum, Clostridium butyricum, Paenibacillus sp.,Veillonella sp., Bifidobacterium, Bacillus subtilis, and Acidaminococcussp.

In some embodiments, an individual microbial strain or combinations ofmicrobial strains from a mammalian microbiome that have been killed(e.g., heat killed). Alternatively, in some embodiments, an individualmicrobial strain or combinations of microbial strains from a mammalianmicrobiome may include cells that are viable or alive.

In some embodiments, one or more microbial strains comprise a viable orliving individual microbial strain or combinations of microbial strains,e.g., from a mammalian microbiome.

In some embodiments, one or more microbial strains comprise a viable orliving individual microbial strain or combinations of microbial strains,e.g., from a mammalian microbiome, as described herein comprises and/oris formulated through use of one or more cell cultures and/orsupernatants or pellets thereof, and/or a powder formed therefrom.

In some embodiments, compositions for use in accordance with the presentdisclosure are pharmaceutical compositions, e.g., for administration(e.g., oral administration) to a mammal (e.g., a human). Pharmaceuticalcompositions typically include an active agent (e.g., individualmicrobial strains or combinations of microbial strains from a mammalianmicrobiome, extracts thereof, and/or components thereof), and apharmaceutically acceptable carrier. Certain exemplary pharmaceuticallyacceptable carriers include, for instance saline, solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like, compatible with pharmaceuticaladministration.

In some embodiments, a pharmaceutical composition for use in accordancewith the present disclosure may include and/or may be administered inconjunction with, one or more supplementary active compounds; in certainembodiments, such supplementary active agents can include ginger,curcumin, probiotics (e.g, probiotic strains of one or more of thefollowing genera: Lactobacillus, Bifidobacterium, Saccharomyces,Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, and/orEscherichia coli (see Fijan, Int J Environ Res Public Health. 2014 May;11(5): 4745-4767, which is incorporated herein by reference); prebiotics(nondigestible food ingredients that help support growth of probioticbacteria, e.g., fructans such as fructooligosaccharides (FOS) andinulins, galactans such as galactooligosaccharides (GOS), dietary fiberssuch as resistant starch, pectin, beta-glucans, and xylooligosaccharides(Hutkins et al., Curr Opin Biotechnol. 2016 February; 37: 1-7, which isincorporated herein by reference) and combinations thereof.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include oral administration. Methods of formulatingsuitable pharmaceutical compositions are known in the art, see, e.g.,Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and thebooks in the series Drugs and the Pharmaceutical Sciences: a Series ofTextbooks and Monographs (Dekker, N.Y.). Oral compositions generallyinclude an inert diluent or an edible carrier. To give but a fewexamples, in some embodiments, an oral formulation may be or comprise asyrup, a liquid, a tablet, a troche, a gummy, a capsule, e.g., gelatincapsules, a powder, a gel, a film, etc.

In some embodiments, pharmaceutically compatible binding agents, and/oradjuvant materials can be included as part of a pharmaceuticalcomposition. In some particular embodiments, a pharmaceuticalcomposition can contain, e.g., any one or more of the following inactiveingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. In some embodiments,the compositions can be taken as-is or sprinkled onto or mixed into afood or liquid (such as water). In some embodiments, a composition thatmay be administered to mammals as described herein may be or comprise aningestible item (e.g., a food or drink) that comprises (e.g., issupplemented) with an individual microbial strain or combinations ofmicrobial strains from a mammalian microbiome, extracts thereof, and/orcomponents thereof.

In some embodiments, a food can be or comprise one or more of bars,candies, baked goods, cereals, salty snacks, pastas, chocolates, andother solid foods, as well as liquid or semi-solid foods includingyogurt, soups and stews, and beverages such as smoothies, shakes,juices, and other carbonated or non-carbonated beverages. In someembodiments, foods are prepared by a subject by mixing in individualmicrobial strains or combinations of microbial strains from a mammalianmicrobiome, extracts thereof, and/or components thereof.

Compositions can be included in a kit, container, pack, or dispenser,together with instructions for administration or for use in a methoddescribed herein.

Those skilled in the art, reading the present disclosure, willappreciate that, in some embodiments, a composition (e.g., apharmaceutical composition) as described herein may be or comprise oneor more cells, tissues, or organisms (e.g., plant or microbe cells,tissues, or organisms) that produce (e.g., have produced, and/or areproducing) a relevant compound.

Those skilled in the art will appreciate that, in some embodiments,technologies for preparing compositions and/or preparations, and/or forpreparing (and particularly for preparing pharmaceutical compositions)may include one or more steps of assessing or characterizing a compound,preparation, or composition, e.g., as part of quality control. In someembodiments, if an assayed material does not meet pre-determinedspecifications for the relevant assessment, it is discarded. In someembodiments, if such assayed material does meet the pre-determinedspecifications, then it continues to be processed as described herein.

In some embodiments, a pharmaceutical composition provided herein canpromote the colonization of an individual microbial strain orcombinations of microbial strains from a mammalian microbiome,particularly microbial strain(s) that have been identified,characterized, or assessed as decreasing the severity or incidence of amammalian disease or condition, in a mammal suffering from or at risk ofthe mammalian disease or condition. In some embodiments, apharmaceutical composition provided herein can attenuate thecolonization of an individual microbial strain or combinations ofmicrobial strains from a mammalian microbiome, particularly microbialstrain(s) that have been identified, characterized, or assessed asincreasing the severity or incidence of a mammalian disease orcondition, in a mammal suffering from or at risk of the mammaliandisease or condition. In some embodiments, a pharmaceutical compositionprovided herein can promote the colonization of an individual microbialstrain or combinations of microbial strains from a mammalian microbiome,particularly microbial strain(s) that have been identified,characterized, or assessed as not affecting the severity or incidence ofthe mammalian disease or condition but have been identified,characterized, or assessed as being capable of outcompeting one or moremicrobial strains that have been identified, characterized, or assessedas increasing the severity or incidence of a mammalian disease orcondition, in a mammal suffering from or at risk of the mammaliandisease or condition.

In some embodiments, each of the one or more microbial strains in acomposition comprises 10¹ to 10¹² colony forming units (CFUs). In someembodiments, each of the one or more microbial strains in a compositioncomprises 10⁶ to 10¹² CFUs. In some embodiments, each of the one or moremicrobial strains in a composition comprises the same number of CFUs. Insome embodiments, some of the one or more microbial strains in acomposition comprises a different number of CFUs.

In some embodiments, a composition comprises a total of 10⁶ to 10¹² ofCFUs.

In some embodiments, a pharmaceutical composition is tailored to aspecific mammal (e.g., a specific human patient) based on that mammal's(e.g., human's) microbiome. In some embodiments, a pharmaceuticalcomposition is specific for a microbiome of an individual mammal (e.g.,human). In some embodiments, a pharmaceutical composition is specificfor microbiomes of a population of mammals (e.g., humans). Populationsof mammals can include, but are not limited to: families, mammals in thesame regional location (e.g., neighborhood, city, state, or country),mammals with the same disease or condition, mammals of a particular ageor age range, mammals that consume a particular diet (e.g., food, foodsource, or caloric intake).

Methods of Treatment

The present disclosure recognizes that compositions described herein canbe useful in the treatment of subjects. Methods provided by the presentdisclosure include methods for the treatment of certain diseases,disorders and conditions. In some embodiments, relevant diseases,disorders and conditions may be or include a neurodegenerative disease,disorder, or condition. In some embodiments, a neurodegenerativedisease, disorder, or condition may be Alzheimer's disease. In someembodiments, relevant diseases, disorders and conditions may be orinclude an ocular neovascular disease, disorder, or condition. In someembodiments, a neurodegenerative disease, disorder, or condition may bediabetic retinopathy, retinopathy of prematurity, age-related maculardegeneration, or glaucoma.

Generally, methods of treatment provided by the present disclosureinvolve administering a therapeutically effective amount of acomposition as described herein alone or in combination with othercompositions and/or treatments to a subject who is in need of, or whohas been determined to be in need of, such treatment.

In some embodiments, methods of treatment provided herein areprophylactic or preventative, e.g., may be administered to subjectsprior to display of significant symptoms and/or to exposure to aparticular expected inducement that is associated with neurodegenerativediseases, disorders, or conditions. In some embodiments, methods oftreatment provided herein are therapeutic, e.g., may be administered tosubjects after development of significant symptoms associated withneurodegenerative diseases, disorders, or conditions.

In some embodiments, provided methods of treatment are administered to asubject that is a mammal, e.g., a mammal that experiences a disease,disorder, or condition as described herein; in some embodiments, asubject is a human or non-human veterinary subject, e.g., an ape, catdog, monkey, or pig.

In many embodiments, treatment involves ameliorating at least onesymptom of a disease, disorder, or condition associated withneurodegenerative diseases, disorders, or conditions. In someembodiments, a method of treatment can be prophylactic.

In some embodiments, the methods can include administration of atherapeutically effective amount of compositions disclosed hereinbefore, during (e.g., concurrently with), or after administration of atreatment that is expected to be associated with neurodegenerativediseases, disorders, or conditions.

In some embodiments, subjects who receive treatment as described hereinmay be receiving and/or may have received other treatment (e.g.,pharmacological treatment/therapy, surgical, etc), for example that maybe intended to treat one or more symptoms or features of a diseasedisorder or condition as described herein (e.g. neurodegenerativediseases, disorders, or conditions), so that provided compositions areadministered in combination with such other therapy (i.e. treatment) totreat the relevant disease, disorder, or condition.

In some embodiments, the compositions described herein can beadministered in a form containing one or more pharmaceuticallyacceptable carriers. Suitable carriers have been described previouslyand vary with the desired form and mode of administration of acomposition. For example, pharmaceutically acceptable carriers caninclude diluents or excipients such as fillers, binders, wetting agents,disintegrators, surface-active agents, glidants, and lubricants.Typically, a carrier may be a solid (including powder), liquid, or anycombination thereof. Each carrier is preferably “acceptable” in thesense of being compatible with other ingredients in the composition andnot injurious to a subject. A carrier can be biologically acceptable andinert (e.g., it permits the composition to maintain viability of thebiological material until delivered to the appropriate site).

Tablets, pills, capsules, troches and the like can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, orange flavoring, or other suitableflavorings. These are for purposes of example only and are not intendedto be limiting.

Oral compositions can include an inert diluent or an edible carrier. Forpurposes of oral therapeutic administration, an active compound can beincorporated with excipients and used in the form of tablets, lozenges,pastilles, troches, or capsules, e.g., gelatin capsules. Oralcompositions can also be prepared by combining a composition of thepresent disclosure with a food. In some embodiments, microbes can beformulated in a food item. Some non-limiting examples of food items tobe used with the methods and compositions described herein include:popsicles, cheeses, creams, chocolates, milk, meat, drinks, pickledvegetables, kefir, miso, sauerkraut, etc. In other embodiments, fooditems can be juices, refreshing beverages, tea beverages, drinkpreparations, jelly beverages, and functional beverages; alcoholicbeverages such as beers; carbohydrate-containing foods such as rice foodproducts, noodles, breads, and pastas; paste products such as fish,hams, sausages, paste products of seafood; retort pouch products such ascurries, food dressed with a thick starchy sauce, and Chinese soups;soups; dairy products such as milk, dairy beverages, ice creams, andyogurts; fermented products such as fermented soybean pastes, fermentedbeverages, and pickles; bean products; various confectionery productsincluding biscuits, cookies, and the like, candies, chewing gums,gummies, cold desserts including jellies, cream caramels, and frozendesserts; instant foods such as instant soups and instant soy-beansoups; and the like. It is preferred that food preparations not requirecooking after admixture with microbial strain(s) to avoid killing anymicrobes. In one embodiment a food used for administration is chilled,for example, iced flavored water. In certain embodiments, the food itemis not a potentially allergenic food item (e.g., not soy, wheat, peanut,tree nuts, dairy, eggs, shellfish or fish). Pharmaceutically compatiblebinding agents, and/or adjuvant materials can be included as part of thecomposition.

In some such embodiments, a composition described herein is administeredto a subject according to a dosing regimen that achieves population ofthe subject's microbiome with administered cells. In some embodiments, acomposition is administered to a subject in a single dose. In someembodiments, a composition is administered to a subject in a pluralityof doses. In some embodiments, a dose of a composition is administeredto a subject twice a day, daily, weekly, or monthly.

In some embodiments, each of the one or more microbial strains in a dosecomprises 10¹ to 10¹² colony forming units (CFUs). In some embodiments,each of the one or more microbial strains in a dose comprises 10⁶ to10¹² CFUs. In some embodiments, each of the one or more microbialstrains in a dose comprises the same number of CFUs. In someembodiments, some of the one or more microbial strains in a dosecomprises a different number of CFUs.

In some embodiments, a dose of one or more microbial strains comprises atotal of 10⁶ to 10¹² CFUs. In some embodiments, a dose of one or moremicrobial strains comprises a total of 10⁷ to 10¹⁰ CFUs. In someembodiments, a dose of one or more microbial strains comprises 5-200billion CFUs. In some embodiments, a dose of one or more microbialstrains comprises 5-50 billion CFUs. In some embodiments, a dose of oneor more microbial strains comprises 5-20 billion CFUs. In someembodiments, a dose of one or more microbial strains comprises 50-100billion CFUs. In some embodiments, a dose of one or more microbialstrains comprises 100-200 billion CFUs.

EXAMPLES

The following examples are provided so as to describe to the skilledartisan how to make and use methods and compositions described herein,and are not intended to limit the scope of the present disclosure.

Example 1: Materials and Methods

Two different constructs for a human APOE4 transgene under the controlof an intestinal promoter (pvha-6::apoE4::tbb-2 UTR) were generated bygene synthesis. The constructs were cloned into the KpnI/SalIrestriction enzyme site of pUC57 plasmid. Human APOE4 protein sequencewas codon optimized for optimal expression in C. elegans. Threesynthetic introns were included within the apoE4 sequence in bothconstructs to avoid gene silencing and optimum expression in C. elegans.The nucleotide sequences of the introns are included in TABLE 1 below.

In one construct, a signal sequence for secretion from C. elegans FLP-1was included in the apoE4 sequence (“Worm 2”). In the other construct,no signal sequence was added to the a oE4 sequence (“Worm 1”).

TABLE 1 Element Sequence Intron 1gtaagtttaaacatatatatactaactaaccctgattatt taaattttcag (SEQ ID NO.: 1)Intron 2 gtaagtttaaacagtteggtactaactaaccatacatatttaaattttcag (SEQ ID NO.: 2) Intron 3gtaagtttaaacatgattttactaactaactaatctgatt taaattttcag (SEQ ID NO.: 3)FLP-1 atgactctgctctaccaagtagggttattactecttgtgg Signalcagctacttataaggtgtcggca (SEQ ID NO.: 4) Sequence

Extrachromosomal array strains were constructed by injecting the eitherWorm 1 or Worm 2 with an expression plasmid and co-injection marker (2ng/μl for pCFJ90, pmyo-2::mCherry). Sequences used for Worm 1 and Worm 2are included in TABLE 2 below. Coding sequences are indicated by capitalletters; non-coding sequences are indicated by small letters.

TABLE 2 WORM 1 vha-6 promoter:actaactgacattaggtgtcacacaaaagaaatcacacactatacatcaaaatatacatcacaagtgagtcaatacaatccgggtgaagctcaagaatggatttcgcagacttettctgctcattggctgcttcgaaaacctgaatagtttatattaaactagtgaaatcgaattcatacaaacctgtttcgattcactacttttcaatcgatggtcaaacgtagaatcaaaaacacgtgtcagaaacacttccaatcatcaaaatgatccatcaattccacteggagcaacaatttcgaagcctgggaatgtgtgtggtgagcacttttggctctggtagagcatgtacctttataggtgcgctctacgcaattcaccagctgaacaatggagttgagcctaatgtaactaaaaatttatttgaatgctttacaaaaatattatttcagatcttcgagatcatgaaaactatcaaacagcagcgccctggagcaatcgagtcgttcccacaatattcaggtgtatatgcaatcgttttagactacatttcggtaagttgctacttcagagataaactgtaattattttaaatttcagcgcaaacgcggaggaaagtctgatcctgttaacaaatacataaatcgtttcctcgctgatctcacggagatattgccagcatgctcaacgttgccagtgatgaatccaagcacagaactgcattaagtatactatttattactcgatacttttgttcacataggttttttaaatcatattttatgcatcatttatcatattcaatgcatcattcatatcatagtcaataaaaaggttgatttctcatgttctggtttcaaatgctgactttggtaaaaagaacgcgtgcctgcctattgcctatcttggcattttctcgataaattttaaaatgtaggttcgatcttatgagatttgtagtcaaaagagctcatatgtattcaggtaggtctggtagcgagaccaacttaatagcatgacaagcattttcaatttgccctggagcgcaattggtifittattcgaaaatcgcacatttctgtttccccataatataaaatttccaggacgatatatattacattettcacaaaatattgcattacagacaccgacaaagaatctccacctgatatgaaaacaatgagccaacaatgttatctgtattgccaccacccacatttcctagtcattcagtatatattgtttcaattgaatcattgcaggtatatatcgaattgaacttgtaaggettcatcttcatttctcaatacatcatccatcattccagagcagctccggccacacaaaaattggtggeggtctgatattgataatcgacttctttgacgtgcctgacggagcagcaaagcggagcactgataagacaatgaagaactaaaaaattgtetteggttttcagtetttagttctgcagcactttatttifigtttctcctatttttccgcattttectaactttctgatgtccatttcaaatgattifigttataaaattgtttaatttcagggcgactaaaacctaccaaaacccataaaaa (SEQ ID NO.: 5) human apoE4:atgAAGGTCCTTTGGGCCGCCCTTCTTGTCACCTTCCTTGCTGGATGCCAAGCTAAGGTTGAGCAAGCTGTTGAAACTGAGCCAGAGCCAGAGCTTCGTCAACAAACTGAGTGGCAATCTGGACAACGTTGGGAGCTTGCTCTTGGACGTTTCTGGGACTACCTTCGTTGGGTTCAAACCCTTTCCGAGCAAGTTCAAGAGGAGCTTCTTTCTTCCCAAGTTACCCAAGAGCTTCGTGCTCTTATGGATGAGACTATGAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGAGCTTAAGGCTTACAAGTCTGAGCTTGAGGAGCAACTTACCCCAGTTGCTGAGGAGACCCGTGCTCGTCTTTCCAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGAGCTTCAAGCTGCTCAAGCTCGTCTTGGAGCTGATATGGAGGATGTTCGTGGACGTCTTGTTCAATACCGTGGAGAGGTTCAAGCTATGCTTGGACAATCTACCGAGGAGCTTCGTGTTCGTCTTGCCTCCCACCTTCGTAAGCTTCGTAAGCGTCTTCTTCGTGACGCTGACGACCTTCAAAAGCGTCTTGCTGTCTACCAAGCTGGAGCTCGTGAGGGAGCTGAGCGTGGACTTTCCGCTATCCGTGAGCGTCTTGGACCACTTGTTGAGCAAGGACGTGTTCGTGCTGCTACCGTCGGATCCCTTGCTGGACAACCACTTCAAGAGCGCGCTCAAGCTTGGGGAGAGCGTCTTCGTGCTCGCATGGAGGAGATGGGATCTCGCACCCGTGATCGTCTTGATGAGGTTAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagGAGCAAGTTGCTGAGGTCCGTGCTAAGCTTGAAGAGCAAGCTCAACAAATCCGTCTTCAAGCTGAGGCTTTCCAAGCTCGTCTTAAGTCTTGGTTCGAGCCACTTGTTGAGGATATGCAACGTCAATGGGCTGGACTTGTTGAGAAGGTCCAAGCCGCTGTCGGAACCTCCGCTGCTCCAGTTCCATCCGATAACCACTAA (SEQ ID NO.: 6) tbb-2 3'UTR:atgcaagatcctttcaagcattcccttcttctctatcactcttctttctttttgtcaaaaaattctctcgctaatttatttgcttttttaatgttattattttatgactifitatagtcactgaaaagtttgcatctgagtgaagtgaatgctatcaaaatgtgattctgtctgatgtactttcacaatctctcttcaattccattttgaagtgctttaaacccgaaaggttgagaaaaatgcgagcgctcaaatatttgtattgtgttcgttgagtgacccaacaaaaagagg aaa (SEQ ID NO.: 7)WORM 2 vha-6 promoter: actaactgacattaggtgtcacacaaaagaaatcacacactatacatcaaaatatacatcacaagtgagtcaatacaatccgggtgaagctcaagaatggatttcgcagacttettctgctcattggctgcttcgaaaacctgaatagtttatattaaactagtgaaatcgaattcatacaaacctgtttcgattcactacttttcaatcgatggtcaaacgtagaatcaaaaacacgtgtcagaaacacttccaatcatcaaaatgatccatcaattccacteggagcaacaatttcgaagcctgggaatgtgtgtggtgagcacttttggctctggtagagcatgtacctttataggtgcgctctacgcaattcaccagctgaacaatggagttgagcctaatgtaactaaaaatttatttgaatgctttacaaaaatattatttcagatcttcgagatcatgaaaactatcaaacagcagcgccctggagcaatcgagtcgttcccacaatattcaggtgtatatgcaatcgttttagactacatttcggtaagttgctacttcagagataaactgtaattattttaaatttcagcgcaaacgcggaggaaagtctgatcctgttaacaaatacataaatcgtttcctcgctgatctcacggagatattgccagcatgctcaacgttgccagtgatgaatccaagcacagaactgcattaagtatactatttattactcgatacttttgttcacataggttttttaaatcatattttatgcatcatttatcatattcaatgcatcattcatatcatagtcaataaaaaggttgatttctcatgttctggtttcaaatgctgactttggtaaaaagaacgcgtgcctgcctattgcctatcttggcattttctcgataaattttaaaatgtaggttcgatcttatgagatttgtagtcaaaagagctcatatgtattcaggtaggtctggtagcgagaccaacttaatagcatgacaagcattttcaatttgccctggagcgcaattggtifittattcgaaaatcgcacatttctgtttccccataatataaaatttccaggacgatatatattacattettcacaaaatattgcattacagacaccgacaaagaatctccacctgatatgaaaacaatgagccaacaatgttatctgtattgccaccacccacatttcctagtcattcagtatatattgtttcaattgaatcattgcaggtatatatcgaattgaacttgtaaggettcatcttcatttctcaatacatcatccatcattccagagcagctccggccacacaaaaattggtggeggtctgatattgataatcgacttctttgacgtgcctgacggagcagcaaagcggagcactgataagacaatgaagaactaaaaaattgtetteggttttcagtetttagttctgcagcactttatttifigtttctcctatttttccgcattttectaactttctgatgtccatttcaaatgattifigttataaaattgtttaatttcagggcgactaaaacctaccaaaacccataaaaa (SEQ ID NO.: 5) human ssapoE4:atgactctgctctaccaagtagggttattactccttgtggcagctacttataaggtgtcggcaAAGGTCCTTTGGGCCGCCCTTCTTGTCACCTTCCTTGCTGGATGCCAAGCTAAGGTTGAGCAAGCTGTTGAAACTGAGCCAGAGCCAGAGCTTCGTCAACAAACTGAGTGGCAATCTGGACAACGTTGGGAGCTTGCTCTTGGACGTTTCTGGGACTACCTTCGTTGGGTTCAAACCCTTTCCGAGCAAGTTCAAGAGGAGCTTCTTTCTTCCCAAGTTACCCAAGAGCTTCGTGCTCTTATGGATGAGACTATGAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGAGCTTAAGGCTTACAAGTCTGAGCTTGAGGAGCAACTTACCCCAGTTGCTGAGGAGACCCGTGCTCGTCTTTCCAAGgtaagtttaaacagtteggtactaactaaccatacatatttaaattttcagGAGCTTCAAGCTGCTCAAGCTCGTCTTGGAGCTGATATGGAGGATGTTCGTGGACGTCTTGTTCAATACCGTGGAGAGGTTCAAGCTATGCTTGGACAATCTACCGAGGAGCTTCGTGTTCGTCTTGCCTCCCACCTTCGTAAGCTTCGTAAGCGTCTTCTTCGTGACGCTGACGACCTTCAAAAGCGTCTTGCTGTCTACCAAGCTGGAGCTCGTGAGGGAGCTGAGCGTGGACTTTCCGCTATCCGTGAGCGTCTTGGACCACTTGTTGAGCAAGGACGTGTTCGTGCTGCTACCGTCGGATCCCTTGCTGGACAACCACTTCAAGAGCGCGCTCAAGCTTGGGGAGAGCGTCTTCGTGCTCGCATGGAGGAGATGGGATCTCGCACCCGTGATCGTCTTGATGAGGTTAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagGAGCAAGTTGCTGAGGTCCGTGCTAAGCTTGAAGAGCAAGCTCAACAAATCCGTCTTCAAGCTGAGGCTTTCCAAGCTCGTCTTAAGTCTTGGTTCGAGCCACTTGTTGAGGATATGCAACGTCAATGGGCTGGACTTGTTGAGAAGGTCCAAGCCGCTGTCGGAACCTCCGCTGCTCCAGTTCCATCCGATAACCACTAA  (SEQ ID NO.: 8) tbb-2 3'UTR:atgcaagatcctttcaagcattcccttcttctctatcactcttctttctttttgtcaaaaaattctctcgctaatttatttgcttttttaatgttattattttatgactifitatagtcactgaaaagtttgcatctgagtgaagtgaatgctatcaaaatgtgattctgtctgatgtactttcacaatctctcttcaattccattttgaagtgctttaaacccgaaaggttgagaaaaatgcgagcgctcaaatatttgtattgtgttcgttgagtgacccaacaaaaagagg aaa (SEQ ID NO.: 7)

TABLE 3 Strain Genotype PD1074 C. elegans wild-type MB1mbEx1(pvha-6::apoE4::tbb-2 UTR + pmyo-2:mCherry) MB2mbEx2(pvha-6::ssapoE4::tbb-2 UTR + pmyo-2:mCherry); SS is signalsequence MB3 mbEx1(pvha-6::apoE4::tbb-2 UTR + pmyo-2:mCherry); dvIs37[myo-3p::GFP::Aβ(3-42) + rol-6(su1006)] MB4 mbEx2(pvha-6::ssapoE4::tbb-2UTR + pmyo-2:mCherry); dvIs37 [myo-3p::GFP::Aβ (3-42) + rol-6(su1006)]CL2331 dvIs37 [myo-3p::GFP::Aβ (3-42) + rol-6(su1006)] MB5mbEx1(pvha-6::apoE4::tbb-2 UTR + pmyo-2:mCherry); dvIs2[pCL12(unc-54/human Aβ peptide 1-42 minigene) + pRF4] MB6mbEx2(pvha-6::ssapoE4::tbb-2 UTR + pmyo-2:mCherry); dvIs2[pCL12(unc-54/human Aβ peptide 1-42 minigene) + pRF4] CL2006 dvIs2[pCL12 (unc-54/human Aβ peptide 1-42 minigene) + pRF4] MB7 mbEx3[unc-119(+);sur-5::UbV-GFP] MB8 mbIs1[F25B3.3::tau352(PHP) + pha-1(+)]MB9 mbEx3 [unc-119(+);sur-5::UbV-GFP]; mbIs1[F25B3.3::tau352(PHP) +pha-1(+)] MB10 mbEx2(pvha-6::ssapoE4::tbb-2 UTR + pmyo-2:mCherry);mbIs1[F25B3.3::tau352(PHP) + pha-1(+)] MB11 mbEx2(pvha-6::ssapoE4::tbb-2UTR + pmyo-2:mCherry); mbEx3 [unc-119(+); sur-5::UbV-GFP] MB12mbEx2(pvha-6::ssapoE4::tbb-2 UTR + pmyo-2:mCherry); mbEx3 [unc-119(+);sur-5::UbV-GFP]; mbIs1[F25B3.3::tau352(PHP) + pha-1(+)] MB13mbEx2(pvha-6::ssapoE4::tbb-2 UTR + pmyo-2:mCherry); mbEx3 [unc-119(+);sur-5::UbV-GFP]; mbIs1[F25B3.3::tau352(PHP) + pha-1(+)]; dvIs2[pCL12(unc-54/human Aβ peptide 1-42 minigene) + pRF4]

Example 2: Exemplary System for Characterizing Microbial Strains ThatAffect a Parameter Associated With Alzheimer's Disease Example 2.1:Alzheimer's Disease

Alzheimer's disease (AD) is the most common cause of dementia. AD ischaracterized by a progressive decline in cognitive functions, includingmemory, language, and cognitive skills. Senile plaques and intracellularneurofibrillary tangles are generally considered hallmark features of ADpathology. Plaques can comprise of aggregates of amyloid-β(Aβ) peptidesof either 40 or 42 amino acids, which can be formed by abnormalprocessing of amyloid precursor protein (APP) by presenilins (PSEN1 andPSEN2). Soluble Aβ oligomers can also cause synaptic dysfunction leadingto neurodegeneration and cognitive disabilities. (Mucke, L., and Selkoe,D. J. (2012). Neurotoxicity of amyloid β-protein: synaptic and networkdysfunction. Cold Spring Harb. Perspect. Med. 2, a006338, which isincorporated herein by reference). The neurofibrillary tangles in AD caninclude hyperphosphorylated tau protein, which is a microtubuleassociated protein in neurons. (Iqbal, K., et al. (2010). Tau inAlzheimer Disease and Related Tauopathies. Curr. Alzheimer Res. 7,656-664, which is incorporated herein by reference). In AD, tau can beabnormally hyperphosphorylated and aggregate into filaments.(Grundke-Iqbal, I., et al. (1986). Abnormal phosphorylation of themicrotubule-associated protein tau (tau) in Alzheimer cytoskeletalpathology. Proc. Natl. Acad. Sci. U.S.A 83, 4913-4917, which isincorporated herein by reference). Because of the strong evidence ofinvolvement of Aβ in AD, several monoclonal antibody-based therapeuticswere developed and tested to target the amyloid plaques. (van Dyck, C.H. (2018). Anti-Amyloid-O Monoclonal Antibodies for Alzheimer's Disease:Pitfalls and Promise. Biol. Psychiatry 83, 311-319, which isincorporated by reference in its entirety). Though studies in standardmammalian animal models showed that interventions that effectivelyprevent or removed Aβ accumulation in animal models, did not improvecognition in human clinical trials.

While about 5% of AD cases appear to have a genetic cause, 95% of thecases are sporadic or late-onset AD with unknown etiology. Less than 1%of AD cases are caused by genetic mutations in genes including APP,PSEN1 and PSEN2. Though several other genes were implicated in AD, oneof the strongest genetic risk factors in AD is apoe. (Lambert, J.-C., etal. (2009). Genome-wide association study identifies variants at CLU andCR1 associated with Alzheimer's disease. Nat. Genet. 41, 1094-1099;Shen, L., and Jia, J. (2016). An Overview of Genome-Wide AssociationStudies in Alzheimer's Disease. Neurosci. Bull. 32, 183-190, each ofwhich is incorporated herein by reference). A lipid/cholesterol carrierapolipoprotein E (APOE) is encoded by apoe. In humans, there are 3 majorprotein variants, termed APOE2, APOE3, and APOE4, that differ from eachother only at two amino acid residues. (Mahley, R. W. (2016).Apolipoprotein E: from cardiovascular disease to neurodegenerativedisorders. J. Mol. Med. Berl. Ger. 94, 739-746, which is incorporatedherein by reference). People carrying polymorphism in apoe, specificallythe apoe4 allele are significantly more likely develop AD, but alsoearly-onset AD, compared to the people who carry either the apoe2 orapoe3 alleles. (Roses, A. D. (1996). Apolipoprotein E alleles as riskfactors in Alzheimer's disease. Annu. Rev. Med. 47, 387-400;Strittmatter, W. J., and Roses, A. D. (1996). Apolipoprotein E andAlzheimer's disease. Annu. Rev. Neurosci. 19, 53-77, each of which isincorporated herein by reference). While APOE2 is thought to be theprotective form of APOE, APOE4 is thought to be the “toxic” form.(Strittmatter and Roses, 1996, which is incorporated herein byreference). APOE4 exacerbates brain changes associated with AD includingincreased levels of amyloid deposits, brain dysfunction andneurodegeneration. (DiBattista, A. M., et al. (2016). Alzheimer'sDisease Genetic Risk Factor APOE-ε4 Also Affects Normal Brain Function.Curr. Alzheimer Res. 13, 1200-1207, which is incorporated herein byreference). Despite the importance of APOE4 in AD, the molecularmechanisms of how APOE4 promotes AD pathogenesis is still notwell-understood. (Kanekiyo, T., et al. (2014). ApoE and Aβ inAlzheimer's disease: accidental encounters or partners? Neuron 81,740-754, which is incorporated by reference in its entirety). APOE4 isthought to contribute to AD pathogenesis via both loss-of-function andgain-of-function mechanisms. (DiBattista, 2016; Zepa, L., et al. (2011).ApoE4-Driven Accumulation of Intraneuronal Oligomerized Aβ42 followingActivation of the Amyloid Cascade In Vivo Is Mediated by a Gain ofFunction. Int. J. Alzheimers Dis. 2011, each of which is incorporatedherein by reference).

Earlier studies suggested that APOE isoforms binds and helps to clearAβ. (Kim, J., et al. (2009). The role of apolipoprotein E in Alzheimer'sdisease. Neuron 63, 287-303, which is incorporated herein by reference).Compared to APOE2 and APOE3, APOE4 was suggested to be less efficient inclearing Aβ (Kim, 2009, which is incorporated herein by reference).However, recent studies suggest that APOE compete with Aβ for uptakethrough apoE receptors. (Verghese, P. B., et al. (2013). APOE influencesamyloid-β (Aβ) clearance despite minimal APOE/Aβ association inphysiological conditions. Proc. Natl. Acad. Sci. U.S.A 110, E1807-1816;Yajima, R., et al. (2015). APOE-isoform-dependent cellular uptake ofamyloid-β is mediated by lipoprotein receptor LR11/SorLA. Biochem.Biophys. Res. Commun. 456, 482-488, each of which is incorporated hereinby reference). While all the isoforms were able to compete for bindingto APOE receptor, APOE4 expressing cells were less efficient in clearingAp. (Verghese, 2013, which is incorporated herein by reference).

Though, the role of APOE4 in Aβ brain pathology has beenwell-documented, the influence of APOE4 in tau pathology has been onlyrecently explored. Using a tauopathy model that overexpress 1N4R humantau containing the P301S mutation, it was shown that ApoE4 exacerbatestau induced neuroinflammation and neurodegeneration phenotypesindependent of Aβ pathology. (Shi, Y., et al. (2017). ApoE4 markedlyexacerbates tau-mediated neurodegeneration in a mouse model oftauopathy. Nature 549, 523-527, which is incorporated herein byreference). The Tau P301S mutation was originally found in human caseswith frontotemporal dementia and degeneration. (Bugiani, O., et al.(1999). Frontotemporal Dementia and Corticobasal Degeneration in aFamily with a P301S Mutation in Tau. J. Neuropathol. Exp. Neurol. 58,667-677, which is incorporated herein by reference). Further, the tauP301S mutant proteins are more favorable substrates for phosphorylationcompared to the wild-type tau. (Alonso, A. del C., et al. (2004).Promotion of hyperphosphorylation by frontotemporal dementia taumutations. J. Biol. Chem. 279, 34873-34881, which is incorporated hereinby reference). Interestingly, the neurofibrillary tangles found in ADare composed primarily of hyperphosphorylated tau. (Iqbal, 2010, whichis incorporated herein by reference). Also, in frontotemporal dementiapatients, the frequency of APOE4 allele is significantly higher(Stevens, M., et al. (1997). Apolipoprotein E gene and sporadic frontallobe dementia. Neurology 48, 1526-1529, which is incorporated herein byreference) and APOE4 carriers also have increased disease severity(Agosta, F., et al. (2009). Apolipoprotein ε4 is associated withdisease-specific effects on brain atrophy in Alzheimer's disease andfrontotemporal dementia. Proc. Natl. Acad. Sci. 106, 2018-2022;Engelborghs, S., et al. (2006). Dose dependent effect of APOE epsilon4on behavioral symptoms in frontal lobe dementia. Neurobiol. Aging 27,285-292, each of which is incorporated herein by reference). Despite theimportance of APOE4 in AD, therapies targeting APOE4 is lacking.(Michaelson, D. M. (2014). APOE ε4: The most prevalent yet understudiedrisk factor for Alzheimer's disease. Alzheimers Dement. J. AlzheimersAssoc. 10, 861-868; Holtzman, D. M., et al. (2012). Apolipoprotein E andApolipoprotein E Receptors: Normal Biology and Roles in AlzheimerDisease. Cold Spring Harb. Perspect. Med. 2, each of which isincorporated herein by reference). Moreover, despite being identified inmore than half of all AD patients, ApoE4 carriers are often excluded inthe clinical trials for AD because of the unpredictability of theirresponse. (Qiu, W. Q., et al. (2013). Angiotensin converting enzymeinhibitors and the reduced risk of Alzheimer's disease in the absence ofapolipoprotein E4 allele. J. Alzheimers Dis. JAD 37, 421-428; Sperling,R., et al. (2012). Amyloid-related imaging abnormalities in patientswith Alzheimer's disease treated with bapineuzumab: a retrospectiveanalysis. Lancet Neurol. 11, 241-249; Farlow, M. R., et al. (1998).Treatment outcome of tacrine therapy depends on apolipoprotein genotypeand gender of the subjects with Alzheimer's disease. Neurology 50,669-677; Risner, M. E., et al. (2006). Efficacy of rosiglitazone in agenetically defined population with mild-to-moderate Alzheimer'sdisease. Pharmacogenomics J. 6, 246-254; each of which is incorporatedherein by reference).

Interestingly, although APOE4 is expressed in the brain, the peripheraltissue expression of APOE4 is high; this raises the possibility thatperipheral APOE4 could contribute to AD pathogenesis. Apart from brain,APOE protein is synthesized primarily in the liver and is involved inlipid transport and cholesterol homeostasis. (Safieh, M., et al. (2019).ApoE4: an emerging therapeutic target for Alzheimer's disease. BMC Med.17, which is incorporated herein by reference). Liver is the primarysite which encounters not only the nutrients but also gutmicrobiome-derived small molecules or metabolites or toxins through theenterohepatic circulation. Thus, dysbiosis in the gut will have profoundeffect on the liver. One possibility is that AD might have a gut-origin:“microbiome-derived materials” might leak into the enterohepaticcirculation and reaches the liver. From liver, the APOE4 might transportthese “microbiome-derived materials” to brain where they could eitherseed amyloid deposits and/or increase neuroinflammation. Recent studieshave suggested that the gut microbiome plays an important role in AD.Significant changes in the microbiome were observed in human AD patientscompared to control populations. However, whether these changes are thecause or consequence of the disease is not known. Though many of themicrobiome components are implicated in either susceptibility orpathogenesis of AD, the molecular mechanisms of such interactionsremains unknown.

Example 2.2: APOE4 Enhances a AP-Induced Paralysis Phenotype

To test whether microbes modulate AD pathogenesis, transgenic C. elegansstrains expressing human APOE4 were developed. These transgenic C.elegans strains expressed human APOE4 in the gut of C. elegans under thecontrol of an intestinal promoter along with a signal sequence thatallows the human APOE4 to be secreted out of the cell [e.g.,mbEx2(pvha-6::ssapoe4)], or without a signal sequence [e.g.,mbEx1(pvha-6::apoe4)]. In C. elegans, there is no liver and the gutperform all the functions that liver typically does. Animals wereadministered with the E. coli OP50 standard laboratory strain. Animalswere monitored from day1 adulthood every alternative day until all wereparalyzed. For each assay, at least 20 animals (as listed in TABLE 4)were recorded. Data from three independent trials was obtained. For eachdata point, mean±s.d is presented in the graph of FIG. 1. Transgenicanimals expressing human APOE4 with or without the signal sequence didnot show any apparent phenotypes (FIG. 1).

Expression of human Aβ1-42 in the C. elegans muscles has been reportedto induce paralysis phenotype. (Link, C. D. (1995). Expression of humanbeta-amyloid peptide in transgenic Caenorhabditis elegans. Proc. Natl.Acad. Sci. U.S.A 92, 9368-9372, which is incorporated herein byreference). To determine whether human APOE4 modulates a paralysisphenotype induced by human Aβ1-42 in the muscles, animals listed inTABLE 4 were analyzed.

TABLE 4 C. elegans Expressed mbEx1(pvha-6::apoe4) APOE4 without signalsequence mbEx2(pvha-6::ssapoe4) APOE4 with signal sequencedvls2(punc-54::Abeta1-42) Aβ1-42 mbEx1(pvha-6::apoe4); APOE4 withoutsignal sequence; Aβ1-42 dvls2(punc-54::Abeta1-42)mbEx2(pvha-6::ssapoe4); APOE4 with signal sequence; Aβ1-42dvls2(punc-54::Abeta1-42)

Expression of APOE4 with a signal sequence enhanced the Aβ-inducedparalysis phenotype. While ˜40% of animals that expressed Aβ wereparalyzed, >90% of animals that expressed both Aβ and APOE4 with asignal sequence were paralyzed by day 8 of adulthood (FIG. 1). However,the paralysis phenotype of animals that expressed Aβ and APOE4 without asignal sequence was similar to that of animals that expressed Aβ byitself (FIG. 1). In contrast, expressing APOE4 by itself, with orwithout the signal sequence, in the absence of Aβ expression did notinduce paralysis phenotype adulthood (FIG. 1).

For the remainder of the studies described herein, a strain thatexpressed APOE4 with a signal sequence, which will be referred tossApoE4, was analyzed.

Example 2.3: Aβ3-42 Conjugated to GFP and Human ssAPOE4 wasSignificantly Increased

Expression of human Aβ₃-42 conjugated with GFP in C. elegans muscles hasbeen reported to cause aggregate formation. (Link, C. D., Fonte, V.,Roberts, C. M., Hiester, B., Silverman, M. A., and Stein, G. H. (2008).The beta amyloid peptide can act as a modular aggregation domain.Neurobiol. Dis. 32, 420-425, which is incorporated herein by reference).To determine whether expression of human ssApoE4 affects Aβ aggregateformation in C. elegans, animals that express human ssApoE4 along withhuman Aβ₃-42 conjugated to GFP were generated. Animals listed in TABLE 5below were administered with the E. coli OP50 standard laboratorystrain.

TABLE 5 C. elegans Expressed GFP::A-Beta (3-42) Aβ3-42ssApoE4::GFP::A-Beta (3-42) APOE4 with signal sequence; Aβ3-42

GFP aggregates in the anterior region of the animal were counted whenthe animals reached adulthood. For each assay, at least 17 animals wererecorded. For each data point, mean±s.d is presented in the graph ofFIG. 2. Compared to animals expressing Aβ3-42, animals expressing Aβ3-42and ssApoE4 had significantly increased GFP aggregates as analyzed usingStudent's t-test, P<0.0001.

Compared to the number of aggregates in the anterior area of animalsthat expressed human Aβ₃-42 conjugated to GFP, the number of aggregatesin animals that expressed both Aβ₃-42 conjugated to GFP and humanssApoE4 was significantly increased (FIG. 2). Interestingly, Aβdeposition in AD patients has been reported to be higher in APOE4carriers in comparison with non-carriers. (Dorey, E., Chang, N., Liu, Q.Y., Yang, Z., and Zhang, W. (2014). Apolipoprotein E, amyloid-beta, andneuroinflammation in Alzheimer's disease. Neurosci. Bull. 30, 317-330,which is incorporated herein by reference).

Example 2.4: UbV-GFP is Stabilized in Animals Expressing Both HumanssAPOE4 and Human tau352 (PHP)

Hyperphosphoryated tau has been reported to be associated with AD.Expression of pseudohyperphosphorylated tau, which mimics AD-relevantmodification, was further reported to induce progressive age-dependentlocomotion defects in C. elegans. (Brandt, R., Gergou, A., Wacker, I.,Fath, T., and Hutter, H. (2009). A Caenorhabditis elegans model of tauhyperphosphorylation: induction of developmental defects by transgenicoverexpression of Alzheimer's disease-like modified tau. Neurobiol.Aging 30, 22-33, which is incorporated herein by reference).

To analyze whether ssAPOE4 modulates tau-induced defects in C. elegans,a transgenic strain that expressed pseudohyperphosphorylated human tauand human ssAPOE4 was generated. Animals of the appropriate genotypeswere administered with the E. coli op50 standard laboratory strain. Noapparent differences locomotion was observed in between the strain thatexpressed pseudohyperphosphorylated human tau and human ssAPOE4 comparedto the strain that expressed the pseudohyperphosphorylated human taustrain alone (data not shown).

Proper proteasomal function is important for cellular function andprevious studies in the field have shown that proteasomal function isimpaired in human AD (Bonet-Costa, V., et al. (2016). The Proteasome andOxidative Stress in Alzheimer's Disease. Antioxid. Redox Signal. 25,886-901; Upadhya, S. C., and Hegde, A. N. (2007). Role of the ubiquitinproteasome system in Alzheimer's disease. BMC Biochem. 8, S12; Oddo, S.(2008). The ubiquitin-proteasome system in Alzheimer's disease. J. Cell.Mol. Med. 12, 363-373; Zheng, Q., et al. (2016). Dysregulation ofUbiquitin-Proteasome System in Neurodegenerative Diseases. Front. AgingNeurosci. 8, each of which is incorporated herein by reference). Todetermine whether ssApoE4 expression affects proteasomal function,animals that carry human ssApoE4 and a marker for impaired proteasomalfunction were generated (TABLE 6). The proteasomal dysfunction markerconsists of a noncleavable ubiquitin that is N-terminally fused to GFP(UbV-GFP).

TABLE 6 C. elegans Expressed UbV-GFP ubiquitin N-terminally fused to GFPssapoe4; APOE4 with signal sequence; ubiquitin N-terminally UbV-GFPfused to GFP Tau352(PHP); Pseudohyperphosphorylated tau protein;ubiquitin N- UbV-GFP terminally fused to GFP ssapoe4; APOE4 with signalsequence; Tau352(PHP); Pseudohyperphosphorylated tau protein; ubiquitinN- UbV-GFP terminally fused to GFP

The number of animals expressing GFP in the gut were counted when theanimals reached adulthood. For each assay, at least 30 animals wererecorded. Data from three independent trials are presented in (FIG. 3).For each data point, mean±s.d is presented in the graph. Compared toanimals that expressed tau352(PHP), animals expressing ssAPOE4 andtau352(PHP) had significantly increased UbV-GFP expression, as analyzedusing Student's t-test, P<0.0001.

Generally, UbV-GFP undergoes proteasomal-dependent degradation, whileimpaired proteostasis causes stabilization of the GFP (see, e.g., FIG.3). Minimal or no GFP expression was observed in animals expressinghuman ssApoE4; however, the UbV-GFP was stabilized in animals expressingboth human ssApoE4 and human pseudohyperphosphorylated human tau (FIG.3). Pseudohyperphosphorylated human tau expression itself did not induceproteasomal stress (FIG. 3). This result suggested that expression ofhuman ssApoE4 and human pseudohyperphosphorylated human tau inducesproteasomal stress.

Hyperphosphorylated Tau has previously been reported to be resistant toproteasomal degradation (Poppek, D., Keck, S., Ermak, G., Jung, T.,Stolzing, A., Ullrich, O., Davies, K. J. A., and Grune, T. (2006).Phosphorylation inhibits turnover of the tau protein by the proteasome:influence of RCAN1 and oxidative stress. Biochem. J. 400, 511-520, whichis incorporated herein by reference) and tau phosphorylation wasreported to modulate proteasomal activity (Ren, Q.-G., Liao, X.-M.,Chen, X.-Q., Liu, G.-P., and Wang, J.-Z. (2007). Effects of tauphosphorylation on proteasome activity. FEBS Lett. 581, 1521-1528;Johnson, G. V. W. (2006). Tau phosphorylation and proteolysis: insightsand perspectives. J. Alzheimers Dis. JAD 9, 243-250, which isincorporated herein by reference). In the C. elegans transgenic strain,human pseudohyperphosphorylated human tau was expressed in the neurons,while human ssApoE4 was expressed under the control of intestinalpromoter along with signal sequences, which allowed it be secreted outof the cell. The induction of UbV-GFP was primarily observed in theintestine of the animals (not shown). The intestine is large prominenttissue in C. elegans, which may mask the induction of UbV-GFP in othertissues. However, induction of UbV-GFP in the intestine provided an easyvisual screening for interventions that might modify the proteasomalfunction.

Example 2.5: Microbial Strains Affect Aβ Paralysis

Animals of the appropriate genotypes were administered with either E.coli OP50 standard laboratory strain or individual microbiome strains.Number of paralyzed animals were recorded on day 4 of adulthood. Datafrom three independent trials are presented. For each data point,mean±s.d is presented in the graph. See Table 1 below for the raw datawith number of animals analyzed for each condition.

To facilitate rapid screening of a microbiome for modulators, a noveltransgenic C. elegans strain that expresses human ssApoE4, human Aβ1-42,human pseudophosphorylated tau and UbV-GFP proteasomal marker wasgenerated. This transgenic strain can be employed for not onlyidentifying interventions that suppress paralysis, but also for agentsthat improve proteasomal function. Further, this model could be used forfinding parameters or features of a biological pathway affect (enhanceor decrease) paralysis. Parameters or features could be small molecules,metabolites, nucleic acids, proteins, lipids, or even microbiomecomponents. Approximately 1400 individual microbial strains from a humanmicrobiome were administered to animals carrying human ssApoE4, humanAβ1-42, human pseudophosphorylated tau and UbV-GFP proteasomal marker. Adegree of paralysis was observed. A group of microbial populations werefound to enhance a paralysis phenotype of animals expressing humanssApoE4, human Aβ1-42, human pseudophosphorylated tau and UbV-GFPproteasomal marker (Table 7; FIG. 4). Enhancement of paralysis wasdependent on the presence of ssAPOE4 because administration of many ofthese bacteria to animals that express human Aβ1-42, humanpseudophosphorylated tau and UbV-GFP proteasomal marker did not enhancethe paralysis phenotype (Table 7; FIG. 4).

TABLE 7 % animal paralyzed (n) in strain expressing % animals paralyzedssAPOE4, Tau352(PHP) (n) in strain expressing Microbial isolate and AβTau352(PHP) and Aβ E. coli OP50 40.6 ± 1.8 (305) 5.5 ± 2.1 (288) E. coliisolate 2 91.6 ± 1.1 (297) 6.3 ± 3.7 (298) E. coli isolate 6 85.6 ± 2.1(298) 5.6 ± 2.9 (305) E. coli isolate 7 83.9 ± 7.1 (299) 5.0 ± 1.7 (301)E. coli isolate 3 51.2 ± 1.1 (299) 5.4 ± 1.6 (299) E. coli isolate 552.9 ± 1.1 (295) 5.3 ± 1.5 (300) E. coli isolate 12 71.9 ± 1.8 (281) 5.1± 2.1 (297) E. coli isolate 15 71.1 ± 2.4 (301) 5.7 ± 2.2 (297) E. coliisolate 8 63.6 ± 1.0 (299) 6.0 ± 1.0 (301) E. coli isolate 21 68.2 ± 2.7(285) 6.0 ± 1.9 (300) E. coli isolate 13 50.7 ± 0.3 (302) 5.2 ± 1.6(286) E. coli isolate 11 51.7 ± 1.8 (302) 5.5 ± 1.4 (289) Escherichiafergusonii isolate 2 68.3 ± 2.4 (297) 5.2 ± 0.2 (290) Escherichiafergusonii isolate 4 76.0 ± 4.2 (304) 5.8 ± 2.0 (292) Escherichiafergusonii isolate 5 82.7 ± 4.4 (304) 5.4 ± 0.7 (299) Escherichiafergusonii isolate 8 63.5 ± 1.8 (296) 6.3 ± 0.6 (304) Escherichiaalbertii isolate 5 57.1 ± 6.1 (291) 5.6 ± 2.1 (302) Escherichia albertiiisolate 3 56.0 ± 8.1 (300) 5.5 ± 0.5 (292) Escherichia albertii isolate8 57.7 ± 10.0 (299) 5.5 ± 1.4 (290) Escherichia albertii isolate 4 67.6± 5.3 (297) 5.5 ± 0.8 (293) Klebsiella oxytoca isolate 1 75.5 ± 5.3(295) 8.7 ± 1.2 (299) Klebsiella oxytoca isolate 8 55.6 ± 4.3 (300) 8.1± 0.6 (308) Klebsiella oxytoca isolate 4 51.7 ± 1.3 (302) 7.5 ± 1.3(305) Klebsiella oxytoca isolate 5 86.7 ± 1.2 (293) 7.6 ± 2.8 (284)Klebsiella pneumoniae isolate 1 70.8 ± 8.6 (300) 9.4 ± 1.6 (299)Klebsiella pneumoniae isolate 3 58.4 ± 3.4 (303) 12.2 ± 0.5 (296)Streptococcus downei 64.8 ± 2.0 (301) 11.6 ± 0.4 (301) Streptococcussanguinis 90.3 ± 2.0 (299) 13.8 ± 1.2 (297) Streptococcus vestibularis75.1 ± 4.4 (297) 13.9 ± 1.4 (297) Streptococcus mitis 80.4 ± 1.4 (305)13.5 ± 0.7 (303) Streptococcus gallolyticus 60.5 ± 5.4 (296) 14.7 ± 0.5(305) Streptococcus anginosus 56.3 ± 3.1 (297) 14.4 ± 1.5 (300)Streptococcus caprinus 77.5 ± 2.9 (298) 12.9 ± 2.2 (302) Streptococcusintermedius 60.8 ± 9.1 (303) 11.9 ± 1.2 (302) Staphylococcus aureus 70.3± 4.0 (303) 13.0 ± 0.3 (307) Staphylococcus epidermis 79.0 ± 1.6 (300)13.0 ± 0.3 (301) Staphylococcus hominis 57.1 ± 8.2 (305) 11.9 ± 1.0(287) Paenisporosarcina sp 69.8 ± 0.4 (288) 12.5 ± 0.6 (296)Paenibacillus sp. 78.1 ± 4.1 (309) 12.5 ± 0.5 (305) Sporosarcina sp.66.8 ± 0.8 (301) 11.7 ± 1.4 (308) Paenibacillus Sp. 64.7 ± 3.1 (287)13.0 ± 0.4 (293) Alcaligenes faecalis isolate 1 74.7 ± 9.9 (297) 7.5 ±3.2 (294) Alcaligenes faecalis isolate 2 57.7 ± 4.1 (297) 6.3 ± 1.1(285) Enterococcus faecium 75.7 ± 6.9 (296) 12.0 ± 1.0 (300)Enterococcus faecalis 73.3 ± 5.6 (304) 13.3 ± 0.5 (309) Deinococcusgrandis 52.9 ± 0.9 (310) 12.5 ± 1.2 (312) Neisseria sp 52.7 ± 3.9 (302)12.6 ± 1.8 (302) Rhodococcus erythropolis 44.6 ± 1.3 (316) 13.3 ± 1.2(316) Corynebacterium amycolatum 56.2 ± 1.5 (301) 11.7 ± 4.3 (292)Actinomyces sp 58.0 ± 2.0 (300) 11.0 ± 0.1 (301) Rothia dentocariosa92.3 ± 1.1 (299) 11.0 ± 3.7 (300) Arcobacter butzlerei 58.3 ± 2.1 (292)13.5 ± 0.7 (304) Citrobacter freundii 58.2 ± 3.9 (301) 11.9 ± 1.5 (301)Acinetobacter baumanni 68.7 ± 1.4 (310) 8.5 ± 2.1 (299) Porphyromonasgingivalis 87.9 ± 5.1 (297) 11.4 ± 0.6 (298) Fusobacterium nucleatum84.4 ± 4.6 (300) 10.9 ± 5.2 (302) Salmonella enterica 64.9 ± 5.0 (291)12.9 ± 1.4 (295) Shigella flexneri 64.5 ± 6.7 (303) 9.8 ± 4.4 (306)Pseudomonas sp. 73.3 ± 8.3 (292) 11.3 ± 4.2 (292) Cardiobacteriumvulvarum 57.9 ± 1.7 (302) 12.9 ± 1.9 (302) Achromabacter oxlosoxidans61.5 ± 3.0 (296) 12.2 ± 1.1 (296)

Example 2.6: Microbial Strains Modulate Aβ3-42::GFP Aggregation

Included in microbial populations that were observed to enhanceparalysis in C. elegans animals expressing human ssApoE4, human Aβ₁₋₄₂,human pseudophosphorylated tau and UbV-GFP proteasomal marker wasPorphyromonas gingivalis (Table 1). P. gingivalis was identified in ADpatient brain and was implicated in neurotoxic tau and amyloiddeposition. (Dominy, S. S., et al. (2019). Porphyromonas gingivalis inAlzheimer's disease brains: Evidence for disease causation and treatmentwith small-molecule inhibitors. Sci. Adv. 5, which is incorporatedherein by reference). Further, P. gingivalis has been reported toincrease ubiquitin load, suggesting a disruption of proteasomalfunction. (Dominy et al., 2019, which is incorporated herein byreference). Oral administration of P. gingivalis was previously shown tobe sufficient to induce brain infection and induction of Aβ deposits(Dominy et al., 2019, which is incorporated herein by reference).

Animals were administered with either E. coli OP50 standard laboratorystrain or individual microbiome strains. GFP aggregates in the anteriorregion of the animal were counted when the animals reached adulthood.GFP aggregates in three animals were counted for each condition. Foreach data point, mean±s.d is presented in the graph.

Administration of P. gingivalis induced increased Aβ₃₋₄₂::GFPaggregates; however, the Aβ₃₋₄₂::GFP aggregates were increasedsignificantly higher in animals expressing human ssApoE4 and Aβ₃₋₄₂::GFPtogether (FIG. 5). This result suggested that the ssApoE4 genotype has adeleterious effect in increasing the incidence AD associated symptoms.Thus, identification of a known microbial population that was previouslyimplicated in AD validates that a transgenic human ssApoE4,pseudophosphorylated tau and Aβ expressing C. elegans platform assay.The results confirm that other microbial populations discovered might befactors that affect AD risk in humans.

Interestingly, several E. coli isolates were found that increased aparalysis phenotype, as well as caused increased Aβ₃₋₄₂::GFP aggregatesin a ssAPOE4 dependent fashion (TABLE 7; FIG. 4; FIG. 5). This isinteresting at least because C. elegans are fed on a standardnon-pathogenic E. coli OP50 strain in the lab. Previous studies haveshown that gram-negative bacterial molecules especially from E. coli areassociated with AD neuropathology. (Zhan, X., Stamova, B., Jin, L.-W.,DeCarli, C., Phinney, B., and Sharp, F. R. (2016). Gram-negativebacterial molecules associate with Alzheimer disease pathology.Neurology 87, 2324-2332, which is incorporated herein by reference). Itis possible that these factors are not expressed or weakly expressed inE. coli OP50 strain or this could suggest strain-specific difference.The 11 strains of E. coli tested were classified into strains that hadeither mild, moderate or severe effect on the paralysis (TABLE 7; FIG.4) or Aβ₃₋₄₂::GFP aggregate phenotype (FIG. 5). These data suggest thatstrain specific differences in microbial populations could contributethe occurrence or severity of AD.

E. fergusonii and E. albertii also showed similar trends to that of E.coli. While some isolates of E. fergusonii and E. albertii increased theparalysis phenotype, others strains had either mild or moderate effects(TABLE 7). This peculiar effect on paralysis was also observed inisolates of Klebsiella oxytoca, Klebsiella pnuemoniae and Alcaligenesfaecalis. This might be a general tendency for other microbialpopulations as well, but because of the number of strains analyzed, thisfeature might be missed. Thus, among other things, the presentdisclosure teaches that individual strains of a particular microbe canhave differential effects on biological phenotype(s), specificallyincluding disease-associated phenotype(s). In some embodiments, thepresent disclosure provides technologies for identifying and/orcharacterizing particular strains, and/or components or combinationsthereof, which may achieve a particular impact on a biologicalphenotype.

Further, microbiome samples from apparently healthy donors wereanalyzed. It is possible that AD patient microbiome samples mightproduce a better trend in identifying strains that might havedeleterious effects. However, using this C. elegans characterizationsystem, a patient microbiome could be assessed for increased ordecreased presence of microbial strains that are associated with or,alternatively affect, disease. Though metagenomic sequencing of patientpopulation could identify the diversity of microbial species present ina particular patient or patient population, these methods cannotidentify strain-level differences in patient samples compared to healthypopulation. The present system fills this gap in identifyingstrain-level differences in patient(s) and/or patient population(s),which may be important for a number of diseases or conditions, includingAD. Thus, this platform could serve us a potential early diagnosticdisease predictor. Thus, among other things, the present disclosureprovides technologies for defining, assessing, and/or detecting,microbes, and/or components or combinations thereof (i.e., microbialsignature(s)), that may be associated with a particular disease states;in some embodiments, such microbial signatures can be detected inpatient sample(s) and, for example, may be useful to diagnose a diseasestate, to monitor impact of a particular therapy with respect to suchdisease state, etc.

Example 2.7: Exemplary Microbial Strains That Affect ATP Production

The Neuro2A cell line was purchased from ATCC and cultured in EMEM mediasupplemented with 10% FBS, and 1% L-glutamine. Cells were maintained at37° C./5% CO₂ incubators. All experiments were carried out using onlypassage 3-7 cells. Neuro2A cells (5×10⁴ cells per well) were plated ontoa 96-well white-walled plate (Corning) and incubated overnight at 37°C./5% CO₂. Each of the 10 bacteria or a combination of all bacteria(CT10) were grown in the following media: Reinforced Clostridial Broth,Peptone Yeast extract Glucose broth, MRS broth and Tryptic Soy Broth.The bacteria were resuspended to 10⁸ CFU in PBS and stored at −80° C.10⁸ CFU of each microbe (referred as samples) was added to 6 wells. Thecombination of all bacteria (CT10) was added to 6 wells at 10⁹ CFU(i.e., 10⁸ CFU of each bacteria). For the control wells, PBS with nobacteria were added. After 16 hours of incubation at 37° C./5% CO₂, 2 μMof Human Amyloid β1-₄₂ was added to all the wells except for controluntreated wells. The cells were incubated for 24 hours at 37° C./5% CO₂.The cells were washed with PBS three-times and 0.05 ml of PromegaCellTiter-Glo and plate was incubated at room temperature shielded fromlight for 1 hour. The luminescence was measured using a microplatereader (Promega discoverer, Promega Corp), indicating the ATP levels.The ATP levels were normalized to protein content, as measured by theBradford Protein Assay kit (ThermoFisher Scientific). 10 μl of sampleswas added to 150 μl of Bradford reagent in clear 96-well plates induplicates and incubated for 5 min in dark at RT and the absorbance wasmeasured at 600 nm using a microplate reader (Promega Discoverer,Promega Corp.). The normalized luminescence was calculated by dividingthe luminescence value by OD protein absorbance value. The average ofthe triplicate wells for each condition was calculated % ATP compared tothe control was calculated.

As shown in FIG. 6, treating Neuro2A cells with 2 μM of Human Amyloidβ₁₋₄₂ (“Mock-treated”) caused significant decrease in ATP production.While treatment of Neuro2A cells with Gluconacetobacter hansenii,Terrisporobacter glycolicus, Coprococcus sp. or Acidaminococcus sp.resulted in significant increase in ATP production compared to theMock-treated cells, a combination of all the bacteria together (CT-10)resulted in further significant increase in the ATP production in thepresence of Human Amyloid β₁₋₄₂.

TABLE 8 Exemplary Microbial Strains Found in Human Gut MicrobiomeBacteroides pectinophilus Exiguobacterium mexicanum Acetobacter spFaecalibacterium prausnitzii Acetobacterium tundrae Faecalitaleacylindroides Achromobacter aegrifaciens Finegoldia magna Achromobacterinsuavis Flavonifractor plautii Achromobacter piechaudii Flintibacterbutyricus Achromobacter xylosoxidans Fusicatenibacter saccharivoransAcidaminococcus fermentans Fusobacterium gonidiaformans Acidaminococcusintestini Fusobacterium mortiferum Acinetobacter baumannii Fusobacteriumnucleatum Acinetobacter junii Fusobacterium ulcerans Actinomyces sp.Fusobacterium varium Agathobacter rectalis Gardnerella vaginalisAgathobaculum butyriciproducens Gemella haemolysans Aggregatibactersegnis Gemella sanguinis Akkermansia muciniphila Gemmiger formicilisAlistipes finegoldii Gluconacetobacter sp Alistipes indistinctusGluconobacter sp Alistipes onderdonkii Gordonibacter pamelaeae Alistipesputredinis Granulicatella adiacens Alistipes shahii Grimontia hollisaeAllisonella histaminiformans Haemophilus parainfluenzae Anaerobaculumhydrogeniformans Harryflintia acetispora Anaerococcus hydrogenalisHelicobacter bilis Anaerococcus octavius Helicobacter bizzozeroniiAnaerococcus prevotii Helicobacter canadensis Anaerococcus tetradiusHelicobacter cinaedi Anaerococcus vaginalis Helicobacter pullorumAnaerofilum agile Helicobacter pylori Anaerofustis stercorihominisHelicobacter winghamensis Anaerosporobacter mobilis Holdemanellabiformis Anaerostipes caccae Holdemania filiformis Anaerostipes hadrusHoldemania massiliensis Anaerostipes rhamnosivorans Hungatella effluviiAnaerotruncus colihominis Hungatella hathewayi Anaerovorax odorimutansIntestinimonas butyriciproducens Arcobacter butzleri Kineothrixalysoides Asaccharobacter celatus Kingella oralis Atopobium parvulumKlebsiella pneumoniae Atopobium vaginae Klebsiella pneumoniae subsp.ozaenae Bacillus cereus Klebsiella pneumoniae subsp. pneumoniae Bacilluscoagulans Klebsiella pneumoniae subsp. rhinoscleromatis Bacilluslicheniformis Klebsiella quasipneumoniae subsp. quasipneumoniae Bacilluspseudomycoides Klebsiella singaporensis Bacillus sonorensis Klebsiellavariicola Bacillus toyonensis Lachnobacterium bovis Bacillus wiedmanniiLachnospira multipara Bacteroides caccae Lachnospira pectinoschizaBacteroides cellulosilyticus Lactobacillus acidophilus Bacteroidesclarus Lactobacillus amylolyticus Bacteroides coprocola Lactobacillusamylovorus Bacteroides coprophilus Lactobacillus antri Bacteroides doreiLactobacillus brevis subsp. Gravesensis Bacteroides eggerthiiLactobacillus buchneri Bacteroides faecis Lactobacillus caseiBacteroides finegoldii Lactobacillus coryniformis subsp. CoryniformisBacteroides fluxus Lactobacillus crispatus Bacteroides fragilisLactobacillus delbrueckii subsp. Bulgaricus Bacteroides intestinalisLactobacillus delbrueckii subsp. indicus Bacteroides massiliensisLactobacillus delbrueckii subsp. Lactis Bacteroides nordii Lactobacillusfermentum Bacteroides oleiciplenus Lactobacillus fructivoransBacteroides ovatus Lactobacillus gasseri Bacteroides plebeiusLactobacillus helveticus Bacteroides salanitronis Lactobacillushilgardii Bacteroides salyersiae Lactobacillus iners Bacteroidesstercoris Lactobacillus jensenii Bacteroides thetaiotaomicronLactobacillus johnsonii Bacteroides uniformis Lactobacillus mucosaeBacteroides vulgatus Lactobacillus oris Bacteroides xylanisolvensLactobacillus paracasei Bacteroides xylanolyticus Lactobacillusparacasei subsp. tolerans Barnesiella intestinihominis Lactobacilluspentosus Bartonella clarridgeiae Lactobacillus plantarum subsp.plantarum Bartonella quintana str. Toulouse Lactobacillus reuteriBifidobacterium adolescentis Lactobacillus rhamnosus Bifidobacteriumangulatum Lactobacillus rogosae Bifidobacterium animalis Lactobacillusruminis Bifidobacterium bifidum Lactobacillus salivarius Bifidobacteriumbreve Lactobacillus ultunensis Bifidobacterium catenulatum Lactobacillusvaginalis Bifidobacterium coryneforme Lactococcus formosensisBifidobacterium dentium Lactococcus garvieae Bifidobacterium faecaleLactococcus lactis subsp. Cremoris Bifidobacterium gallicum Lactococcuslactis subsp. lactis Bifidobacterium longum Lactonifactor longoviformisBifidobacterium longum subsp. infantis Laribacter hongkongensisBifidobacterium longum subsp. longum Lautropia mirabilis Bifidobacteriumlongum subsp. suis Leptotrichia buccalis Bifidobacteriumpseudocatenulatum Leptotrichia hofstadii Bifidobacterium pseudolongumLeuconostoc lactis Bifidobacterium stercoris Leuconostoc mesenteroidessubsp. Cremoris Bilophila wadsworthia Listeria grayi Bittarellamassiliensis Listeria monocytogenes Blautia coccoides Longicatenacaecimuris Blautia faecis Marvinbryantia formatexigens Blautiaglucerasea Megamonas funiformis Blautia hansenii Megamonas rupellensisBlautia hydrogenotrophica Megasphaera elsdenii Blautia luti Megasphaeraindica Blautia obeum Megasphaera micronuciformis Blautia productaMegasphaera paucivorans Blautia schinkii Methanobrevibacter smithiiBlautia stercoris Methanomassiliicoccus luminyensis Blautia wexleraeMethanosphaera stadtmanae Bradyrhizobium japonicum Methylobacteriumradiotolerans Burkholderia ambifaria Mitsuokella jalaludiniiBurkholderia cenocepacia Mitsuokella multacida Burkholderia glumaeMobiluncus mulieris Burkholderia multivorans Mogibacterium timidumBurkholderia plantarii Mogibacterium vescum Butyricicoccus faecihominisMoraxella catarrhalis Butyricicoccus pullicaecorum Morganella morganiisubsp. morganii Butyricimonas faecihominis Murdochiella asaccharolyticaButyricimonas paravirosa Mycobacterium abscessus Butyricimonas virosaMycobacterium tuberculosis Butyrivibrio crossotus Mycoplasma hominisCampylobacter coli Neisseria cinerea Campylobacter concisus Neisseriaflavescens Campylobacter curvus Neisseria macacae Campylobacter gracilisNeisseria mucosa Campylobacter hominis Neisseria sicca Campylobacterjejuni subsp. Jejuni Neisseria subflava Campylobacter showae Nitrobacterhamburgensis Campylobacter upsaliensis Nitrobacter winogradskyiCandidatus Dorea massiliensis Odoribacter laneus Candidatus Stoquefichusmassiliensis Odoribacter splanchnicus Capnocytophaga gingivalisOlsenella profusa Capnocytophaga sputigena Olsenella scatoligenesCardiobacterium hominis Olsenella uli Catenibacterium mitsuokaiOribacterium sinus Catonella morbi Oscillibacter ruminantium Cedecealapagei Oscillibacter valericigenes Citrobacter amalonaticusOscillospira guilliermondii Citrobacter freundii Oxalobacter formigenesCitrobacter koseri Paenibacillus jamilae Citrobacter youngaePaenibacillus kribbensis Clostridium acetobutryicum Paenibacillusriograndensis Clostridium aerotolerans Paeniclostridium sordelliiClostridium aldenense Parabacteroides distasonis Clostridium aminophilumParabacteroides goldsteinii Clostridium aminovalericum Parabacteroidesgordonii Clostridium amygdalinum Parabacteroides johnsonii Clostridiumasparagiforme Parabacteroides merdae Clostridium baratii Paraprevotellaclara Clostridium bartlettii Paraprevotella xylaniphila Clostridiumbeijerinckii Parasutterella excrementihominis Clostridium bifermentansParasutterella secunda Clostridium bolteae Parvimonas micra Clostridiumbutyricum Pediococcus acidilactici Clostridium celerecrescensPediococcus pentosaceus Clostridium cf. saccharolyticum Peptoniphilusduerdenii Clostridium citroniae Peptoniphilus grossensis Clostridiumclariflavum Peptoniphilus harei Clostridium clostridioformePeptoniphilus indolicus Clostridium cocleatum Peptostreptococcusanaerobius Clostridium colinum Phascolarctobacterium faecium Clostridiumdifficile Phascolarctobacterium succinatutens Clostridiumglycyrrhizinilyticum Porphyromonas asaccharolytica Clostridium hathewayiPorphyromonas endodontalis Clostridium herbivorans Porphyromonasgingivalis Clostridium hiranonis Prevotella bivia Clostridium hylemonaePrevotella buccae Clostridium innocuum Prevotella copri Clostridiumlactatifermentans Prevotella disiens Clostridium lavalense Prevotellamarshii Clostridium leptum Prevotella melaninogenica Clostridiummethoxybenzovorans Prevotella nigrescens Clostridium methylpentosumPrevotella pallens Clostridium nexile Prevotella salivae Clostridiumorbiscindens Prevotella stercorea Clostridium oroticum Prevotellatannerae Clostridium perfringens Prevotella timonensis Clostridiumpolysaccharolyticum Propionibacterium acnes Clostridium propionicumPropionibacterium avidum Clostridium ramosum Propionibacteriumnamnetense Clostridium rectum Proteus mirabilis Clostridiumsaccharogumia Proteus penneri Clostridium saccharolyticum Providenciaalcalifaciens Clostridium sardiniense Providencia rettgeri Clostridiumsaudii Providencia rustigianii Clostridium scindens Providencia stuartiiClostridium sordellii Pseudoflavonifractor capillosus Clostridiumsphenoides Ralstonia sp. Clostridium spiroforme Robinsoniella peoriensisClostridium sporogenes Roseburia cecicola Clostridium sticklandiiRoseburia faecis Clostridium straminisolvens Roseburia hominisClostridium symbiosum Roseburia intestinalis Clostridium tertiumRoseburia inulinivorans Clostridium thermocellum Rothia dentocariosaClostridium xylanolyticum Ruminococcus albus Clostridium xylanovoransRuminococcus bromii Collinsella aerofaciens Ruminococcus callidusCollinsella intestinalis Ruminococcus faecis Collinsella stercorisRuminococcus gnavus Collinsella tanakaei Ruminococcus lactarisCoprobacillus cateniformis Ruminococcus obeum Coprobacter fastidiosusRuminococcus torques Coprococcus catus Ruthenibacterium lactatiformansCoprococcus comes Sarcina ventriculi Coprococcus eutactus Sellimonasintestinalis Corynebacterium ammoniagenes Senegalimassiiia anaerobiaCorynebacterium matruchotii Shigella boydii Corynebacteriumpseudogenitalium Shigella dysenteriae Corynebacterium tuberculostearicumShigella flexneri Deinococcus radiodurans Shigella sonnei Dermabacterhominis Slackia faecicanis Desuifotomaculum guttoideum Slackiaisoflavoniconvertens Desulfovibrio legallis Slackia piriformisDesulfovibrio piger Solobacterium moorei Dialister invisusStaphylococcus caprae Dialister microaerophilus Staphylococcusepidermidis Dialister succinatiphilus Staphylococcus hominis subsp.Hominis Dielma fastidiosa Staphylococcus lugdunensis Doreaformicigenerans Staphylococcus warneri Dorea longicatena Streptococcusagalactiae Dysgonomonas mossii Streptococcus anginosus Edwardsiellatarda Streptococcus anginosus subsp. whileyi Eggerthella lentaStreptococcus australis Eggerthella sinensis Streptococcus bovisEikenella corrodens Streptococcus constellatus subsp. constellatusEisenbergiella tayi Streptococcus equinus Enhydrobacter aerosaccusStreptococcus gallolyticus subsp. pasteuri Enterobacter aerogenesStreptococcus gallolyticus subsp. pasteurianus Enterobacter asburiaeStreptococcus gordonii Enterobacter cancerogenus Streptococcus gordoniistr. Challis Enterobacter cloacae Streptococcus infantarius Enterobacterhormaechei Streptococcus infantarius subsp. coli Enterobacter kobeiStreptococcus infantarius subsp. Infantarius Enterobacter ludwigiiStreptococcus infantis Enterobacter xiangfangensis Streptococcuslactarius Enterococcus asini Streptococcus lutetiensis Enterococcusavium Streptococcus mutans Enterococcus casseliflavus Streptococcusparasanguinis Enterococcus durans Streptococcus pasteurianusEnterococcus faecalis Streptococcus pleomorphus Enterococcus faeciumStreptococcus rubneri Enterococcus gallinarum Streptococcus salivariusEnterococcus hirae Streptococcus salivarius subsp. salivariusEnterococcus mundtii Streptococcus sanguinis Enterococcus raffinosusStreptococcus thermophilus Enterococcus raffinosus Streptococcusvestibularis Erysipelotrichaceae bacterium Subdoligranulum variabileEscherichia albertii Succinatimonas hippei Escherichia coli Sutterellaparvirubra Escherichia fergusonii Sutterella stercoricanis Eubacteriumbiforme Sutterella wadsworthensis Eubacterium callanderiTerrisporobacter glycolicus Eubacterium contortum Turicibacter sanguinisEubacterium cylindroides Ureaplasma parvum Eubacterium desmolansVagococcus penaei Eubacterium dolichum Varibaculum cambrienseEubacterium eligens Veillonella sp. Eubacterium hadrum Veillonelladispar Eubacterium hallii Veillonella parvula Eubacterium infirmumVeillonella rogosae Eubacterium limosum Veillonella tobetsuensisEubacterium oxidoreducens Vibrio cholerae Eubacterium ramulus Vibriofurnissii Eubacterium rectale Vibrio mimicus Eubacterium ruminantiumVictivallis vadensis Eubacterium saburreum Weissella cibaria Eubacteriumsiraeum Weissella confusa Eubacterium sulci Weissella paramesenteroidesEubacterium tortuosum Xenorhabdus nematophila Eubacterium ventriosumYersinia enterocolitica subsp. Palearctica Eubacterium xylanophilumYersinia pseudotuberculosis Eubacterium yurii subsp. Margaretiae

Example 3: Exemplary System for Characterizing Microbial Strains ThatAffect HIF Pathway Example 3.1: HIF Pathway

Hypoxia inducible factor (HIF) pathway mediates a diversity of metabolicand physiological adaptations to reduced oxygen levels in cells.Activation of the HIF pathway promotes erythropoiesis and angiogenesisto reduce the cellular requirement for oxygen. Though the HIF pathway isimportant for cellular stress response, constitutive activation of HIF-1leads to neovascularization in conditions such as diabetic retinopathy,retinopathy of prematurity, age-related macular degeneration, andglaucoma. Thus, developing modulators of HIF pathway is essential forthe treatment of ocular neovascular diseases.

The HIF-1 pathway consists of the HIF transcription factor and thenegative regulator, the prolyl hydroxylase EGLN. EGLN functions asoxygen sensor and in the presence of oxygen it hydroxylates the HIFα-subunit (HIFα). Hydroxylation of HIFα leads to binding to vonHippel-Lindau (VHL) E3 ubiquitin ligase, among other factors, whichpromote HIFα degradation (see FIG. 7). Under low oxygen conditions, HIFαprotein is stabilized and it promotes transcriptional activation ofgenes required for adaptation to low oxygen levels.

Example 3.2: Constitutively Active HIF-1 Results in Defect in Egg Laying

In C. elegans, egl-9 encodes the EGLN homolog. In egl-9 loss-of-function(egl-9 lf) C. elegans mutants, HIF-1, which is the homolog of HIF1α,protein levels are stabilized. Thus, the HIF1 protein is constitutivelyactive leading to continuous activity of HIF-1 transcriptional targetgenes. To identify microbes that regulate HIF-1 pathway, egl-9loss-of-function (egl-9 lf) C. elegans mutants were analyzed. egl-9 lfmutant C. elegans have constitutively active HIF-1, which results inthem being defective in laying eggs. Therefore, they become bloated witheggs as adults.

Example 3.3: Microbial Strains Affect HIF-1 Induced Egg Laying Defects

HIF-1 modulators were identified by screening individual bacterialstrains for the ability to suppress the egg laying defect of egl-9 lfmutant. While wildtype animals lay 8±2 (n=30) eggs per hour, egl-9 lfmutants lay 2±1 (n=30) eggs/hour. The egl-9 lf C. elegans mutants wereadministered with each individual microbe and the respective egg-layingrate was measured. In this assay, it was found that Gluconacetobacterspp and Bifidobacterium spp significantly enhanced the egg-laying rateof egl-9 lf C. elegans mutants to 11±2 (n=25) and 8±2 (n=29)respectively (see Table 9). This example demonstrates that microbialstrains, such as those in Table 9, can modulate HIF-1 and a HIF-1pathway, and can be used to ameliorate conditions and diseasesassociated with an alteration of a HIF-1 pathway.

TABLE 9 egl-9 lf C. elegans Egg-laying Number of administered with rateanimals tested E. coli OP50-1 2 ± 1 30 Gluconacetobacter hansenii 11 ±2  25 Terrisporobacter glycolicus 3 ± 1 32 Coprococcus sp. 1 ± 1 28 L.plantarum 2 ± 2 29 Clostridium butyricum 3 ± 1 31 Paenibacillusbarengoltzii 1 ± 2 27 Veillonella atypica 1 ± 1 30 Bifidobacterium 8 ± 229 Bacillus subtilis 4 ± 2 28 Acidaminococcus sp 3 ± 2 31

OTHER EMBODIMENTS

It is to be appreciated by those skilled in the art that variousalterations, modifications, and improvements to the present disclosurewill readily occur to those skilled in the art. Such alterations,modifications, and improvements are intended to be part of the presentdisclosure, and are intended to be within the spirit and scope of theinvention. Accordingly, the foregoing description and drawing are by wayof example only and any invention described in the present disclosure iffurther described in detail by the claims that follow.

Those skilled in the art will appreciate typical standards of deviationor error attributable to values obtained in assays or other processes asdescribed herein. The publications, websites and other referencematerials referenced herein to describe the background of the inventionand to provide additional detail regarding its practice are herebyincorporated by reference in their entireties.

It is to be understood that while embodiments of the invention have beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1.-62. (canceled)
 63. A composition comprising one or more microbialstrains listed in TABLE
 8. 64. The composition of claim 63, comprisingtwo or more microbial strains listed in TABLE
 8. 65. The composition ofclaim 63, comprising five or more microbial strains listed in TABLE 8.66. The composition of claim 63, comprising 10 or more microbial strainslisted in TABLE
 8. 67. A composition comprising Gluconacetobacterhansenii, Terrisporobacter glycolicus, Coprococcus sp., L. plantarum,Clostridium butyricum, Paenibacillus sp., Veillonella sp.,Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or acombination thereof.
 68. The composition of claim 67, comprising atleast two microbial strains selected from the group consisting ofGluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcussp., L. plantarum, Clostridium butyricum, Paenibacillus sp., Veillonellasp., Bifidobacterium, Bacillus subtilis, and Acidaminococcus sp.
 69. Thecomposition of claim 67, comprising at least five microbial strainsselected from the group consisting of Gluconacetobacter hansenii,Terrisporobacter glycolicus, Coprococcus sp., L. plantarum, Clostridiumbutyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium, Bacillussubtilis, and Acidaminococcus sp.
 70. The composition of claim 67,comprising Gluconacetobacter hansenii, Terrisporobacter glycolicus,Coprococcus sp., L. plantarum, Clostridium butyricum, Paenibacillus sp.,Veillonella sp., Bifidobacterium sp., Bacillus subtilis, andAcidaminococcus sp.
 71. The composition of claim 67, wherein thecomposition is a pharmaceutical composition.
 72. The composition ofclaim 67, wherein the composition is an ingestible item.
 73. A method oftreating a disease or condition in a subject, comprising administeringto a subject in need a composition comprising the composition of claim67.
 74. The method of claim 73, wherein the disease or condition is aneurodegenerative disease or disorder.
 75. The method of claim 73,wherein the disease or condition is Alzheimer's disease.
 76. The methodof claim 73, wherein the disease or condition is associated with analtered or defective HIF-1 pathway.
 77. The method of claim 73, whereinthe disease or condition is an ocular neovascular disease or disorder.78. The method of claim 73, wherein the disease or condition is diabeticretinopathy, retinopathy of prematurity, age-related maculardegeneration, or glaucoma.
 79. Use of a composition of claim 67 in thetreatment of a disease or condition in a subject.
 80. The use of claim79, wherein the disease or condition is a neurodegenerative disease ordisorder.
 81. The use of claim 79, wherein the disease or condition isAlzheimer's disease.
 82. The use of claim 79, wherein the disease orcondition is associated with an altered or defective HIF-1 pathway. 83.The use of claim 79, wherein the disease or condition is an ocularneovascular disease or disorder.
 84. The use of claim 79, wherein thedisease or condition is diabetic retinopathy, retinopathy ofprematurity, age-related macular degeneration, or glaucoma.